Methods of using regenerative cells to promote wound healing

ABSTRACT

Cells present in adipose tissue are used to promote wound healing in a patient. Methods of treating patients include processing adipose tissue to deliver a concentrated amount of regenerative cells obtained from the adipose tissue to a patient. The methods may be practiced in a closed system so that the regenerative cells are not exposed to an external environment prior to being administered to a patient. Accordingly, in a preferred method, cells present in adipose tissue are placed directly into a recipient along with such additives necessary to promote, engender or support a therapeutic benefit.

RELATED APPLICATIONS

This application is a divisional application of and claims priorityunder 35 U.S.C. §120 to U.S. application Ser. No. 13/360,022, filed onJan. 27, 2012, entitled “METHODS OF USING REGENERATIVE CELLS TO PROMOTEWOUND HEALING,” which is a divisional application of and claims priorityunder 35 U.S.C. §120 to U.S. application Ser. No. 10/884,860, filed onJul. 1, 2004, entitled “METHODS OF USING REGENERATIVE CELLS TO PROMOTEWOUND HEALING,” which is a continuation-in-part application of, andclaims priority to U.S. application Ser. No. 10/316,127, filed on Dec.9, 2002, entitled “SYSTEMS AND METHODS FOR TREATING PATIENTS WITHPROCESSED LIPOASPIRATE CELLS,” which claims the benefit of U.S.Provisional Application No. 60/338,856, filed Dec. 7, 2001. The contentsof the aforementioned applications are expressly incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to regenerative cells derived from awide variety of tissues, and more particularly, to adipose-derivedregenerative cells (e.g., stem and/or progenitor cells), methods ofusing adipose-derived regenerative cells, compositions containingadipose-derived regenerative cells, and systems for preparing and usingadipose-derived regenerative cells which are used to promote woundhealing, e.g., wounds resulting from diabetes, chronic peripheralvascular disease and obesity

2. Description of Related Art

Approximately five million Americans suffer from chronic open soresoften due to limited blood flow which can slow the body's own healingprocess. Normally, injury to the skin sets into motion a complex cascadeof events which ultimately results in wound healing, namely theformation of the neodermis and re-epithelialization. This complexsequence of events includes a symphony of interaction between local,regional and systemic growth factors, cytokines, as well as cellularparticipants, including mesenchymal stem cells. Inadequate and/orincomplete wound healing, however, can lead to significant individualmorbidity including a profound reduction in quality of life. Sores canbecome seriously infected, gangrenous and in some cases requireamputation. For example, the primary cause of non-traumatic amputationdue to inadequate wound healing is diabetes. There are nearly 16 milliondiabetics in the United States. More than 67,000 patients with diabetesrequire surgical amputation each year, necessitating long and costlyrehabilitation. For many, mobility and independence are severelyaffected, permanently altering their quality of life. At its extreme,poor wound healing can serve as the root cause of death.

For patients who have chronic wounds that are difficult to heal, basicmedical and surgical care is essential, but not always enough. Currenttherapies for wound healing include removal of unhealthy tissue, use ofgrowth factors, hyperbaric (high-pressure) oxygen treatment, advancedwound dressings, antibiotic therapy, conventional wound dressings,nutrition counseling, education/prevention, surgery, and physicaltherapy. For example, manipulation of a single or a combination ofparticipating molecules, including but not limited to PDGF-BB and bFGF,can be used to augment natural wound healing. In addition, applicationof “engineered” cellular products (Apligraf® and Dermagraft®) as a wayof engendering a more physiologic pattern of growth factor expressioncan also been used. The implementation of therapies like Regranex, aplatelet-derived growth factor which stimulates healing, has also provento be beneficial for wound care in appropriate patients.

The regenerative cellular approach has also enjoyed relative successcompared to single or combination molecular therapies described above.Regenerative medicine harnesses, in a clinically targeted manner, theability of stem cells (i.e., the unspecialized master cells of the body)to renew themselves indefinitely and develop into mature specializedcells. Broad application of regenerative cellular therapy, however, hasbeen hindered by questions of cellular retention and the high costsassociated with extensive ex vivo manipulation. However, although stemcell populations have been shown to be present in one or more of bonemarrow, skin, muscle, liver and brain (Jiang et al., 2002b; Alison,1998; Crosby and Strain, 2001), their frequency in these tissues is low.For example, mesenchymal stem cell frequency in bone marrow is estimatedat between 1 in 100,000 and 1 in 1,000,000 nucleated cells (D'Ippolitoet al., 1999; Banfi et al., 2001; Falla et al., 1993). Similarly,extraction of ASCs from skin involves a complicated series of cellculture steps over several weeks (Toma et al., 2001) and clinicalapplication of skeletal muscle-derived stem cells requires a two tothree week culture phase (Hagege et al., 2003). Thus, any proposedclinical application of stem cells from such tissues requires increasingcell number, purity, and maturity by processes of cell purification andcell culture.

Although cell culture steps may provide increased cell number, purity,and maturity, they do so at a cost. This cost can include one or more ofthe following technical difficulties: loss of cell function due to cellaging, loss of potentially useful non-stem cell populations, delays inpotential application of cells to patients, increased monetary cost, andincreased risk of contamination of cells with environmentalmicroorganisms during culture. Recent studies examining the therapeuticeffects of bone-marrow derived ASCs have used essentially whole marrowto circumvent the problems associated with cell culturing (Horwitz etal., 2001; Orlic et al., 2001; Stamm et al., 2003; Strauer et al.,2002). The clinical benefits, however, have been suboptimal, an outcomealmost certainly related to the limited ASC dose and purity inherentlyavailable in bone marrow.

Recently, adipose tissue has been shown to be a source of stem cells(Zuk et al., 2001; Zuk et al., 2002). Adipose tissue (unlike marrow,skin, muscle, liver and brain) is comparably easy to harvest inrelatively large amounts with low morbidity (Commons et al., 2001; Katzet al., 2001b). Suitable methods for harvesting adipose derived stemcells, however, are lacking in the art. The existing methods suffer froma number of shortcomings. For example, the existing methods lack partialor full automation, a partial or completely closed system, disposabilityof components, etc.

Given the therapeutic potential of adipose derived stem cells for woundhealing, there exists a need in the art for a method for harvestingcells from adipose tissue that produces a population of adult stem cellswith increased yield, consistency and/or purity and does so rapidly andreliably with a diminished or non-existent need for post-extractionmanipulation.

SUMMARY OF THE INVENTION

The present invention relates to regenerative cells, e.g., adult stemand progenitor cells, that can be used to promote wound healing. Thepresent invention also relates to systems and methods for separating andconcentrating regenerative cells from tissue, e.g., adipose tissue. Thepresent invention further relates to compositions of regenerative cellsfor wound healing applications. Accordingly, in a general embodiment,the present invention is directed to compositions, methods, and systemsfor using regenerative cells derived from tissue that are placeddirectly into a recipient along with such additives necessary topromote, engender, or support a therapeutic wound healing benefit.

In specific embodiments, the present invention is directed to methodsfor promoting wound healing, by administering a concentration ofregenerative cells. The regenerative cells may be comprised of, e.g.,stem cells, progenitor cells or combination thereof. In certainembodiments, administration of multiple doses of regenerative cells maybe needed to derive a therapeutic benefit. In addition, additives suchas one or more growth factors may be administered with the regenerativecells. In a preferred embodiment, the regenerative cells areadministered with angiogenic growth factors alone or in combination withother additives. The regenerative cells may also be administered withone or more immunosuppressive drugs.

The routes of administration for the regenerative cells are known in theart and include injection into a subcutaneous, dermal or intramuscularin site distant from the injury or localized vascular delivery using acatheter based mechanism. The cells may be administered to, for examplethe patient's vasculature. The cells may also be administered via ascaffold, e.g., a resorbable scaffold, or a bandage known in the art.

Prior to administration to a patient, the regenerative cells may begrown in cell culture to, for example, promote differentiation towards aepithelial and/or endothelial phenotype. The cell culture may beperformed on a scaffold material, e.g., a resorbable scaffold, togenerate a two or three dimensional construct that can be placed on orwithin the patient. Prior to administration to a patient, the cellscould also be modified by gene transfer such that expression of one ormore genes, e.g., an angiogenic gene, in the modified regenerative cellsis altered.

The present invention also relates to highly versatile systems andmethods capable of separating and concentrating regenerative cells,e.g., stem and progenitor cells, from a given tissue, that are suitablefor re-infusion into a subject. In a preferred embodiment, the system isautomated. The system of the present invention generally includes one ormore of a collection chamber, a processing chamber, a waste chamber, anoutput chamber and a sample chamber. The various chambers are coupledtogether via one or more conduits such that fluids containing biologicalmaterial may pass from one chamber to another in a closed, sterilefluid/tissue pathway. In certain embodiments, the waste chamber, theoutput chamber and the sample chamber are optional. In one embodiment,the entire procedure from tissue extraction through processing andplacement of the device into the recipient would all be performed in thesame facility, indeed, even within the same room of the patientundergoing the procedure.

Accordingly, in one embodiment, a method of promoting wound healing in apatient includes steps of: a) providing a tissue removal system; b)removing adipose tissue from a patient using the tissue removal system,the adipose tissue having a concentration of stem cells; c) processingat least a part of the adipose tissue to obtain a concentration ofregenerative cells other than the concentration of regenerative cells ofthe adipose tissue before processing; and d) administering theregenerative cells to a patient without removing the regenerative cellsfrom the tissue removal system before being administered to the patient.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is an illustration of a system for separating regenerative cellsfrom tissue which includes one filter assembly.

FIG. 2 is an illustration of a system similar to FIG. 1 having aplurality of filter assemblies in a serial configuration.

FIG. 3 is an illustration of a system similar to FIG. 1 having aplurality of filter assemblies in a parallel configuration.

FIG. 4 is an illustration of a system for separating regenerative cellsfrom tissue which includes a centrifuge chamber.

FIG. 5 is a sectional view of a collection chamber including a prefixedfilter utilized in a system for separating regenerative cells fromtissue.

FIG. 6 is a sectional view of a processing chamber of a system forseparating regenerative cells from tissue utilizing a percolativefiltration system.

FIG. 7 is a sectional view of a processing chamber of a system forseparating regenerative cells utilizing a centrifuge device forconcentrating the regenerative cells.

FIG. 8 is another sectional view of the processing chamber of FIG. 7.

FIGS. 9.1, 9.2 and 9.3 illustrate an elutriation component in use withthe system of the invention.

FIG. 10 is an illustration of a system for separating regenerative cellsfrom tissue utilizing vacuum pressure to move fluids through the system.A vacuum system can be constructed by applying a vacuum pump or vacuumsource to the outlet of the system, controlled at a predetermined rateto pull tissue and fluid through, using a system of stopcocks, vents,and clamps to control the direction and timing of the flow.

FIG. 11 is an illustration of a system for separating regenerative cellsfrom tissue utilizing positive pressure to move fluids through thesystem. A positive pressure system uses a mechanical means such as aperistaltic pump to push or propel the fluid and tissue through thesystem at a determined rate, using valves, stopcocks, vents, and clampsto control the direction and timing of the flow.

FIG. 12A illustrates a filtration process in which the feed stream offluid flows tangentially to the pores of the filter. FIG. 12Billustrates a filtration process in which the feed stream of fluid flowsperpendicular to the pores of the filter.

FIG. 13 is an illustration of an exemplary disposable set for a systemof the invention.

FIG. 14 is an illustration of an exemplary re-usable component for asystem of the invention.

FIG. 15A is an illustration of an exemplary device of the inventionassembled using the disposable set of FIG. 13 and a re-usable componentof FIG. 14.

FIG. 15B is a flowchart depicting exemplary pre-programmed steps,implemented through a software program, that control automatedembodiments of a system of the present invention. Two alternativeprocessing parameters are shown indicating the versatility of thesystem.

FIGS. 16A and 16B depict the expression of VEGF (5A) and PIGF (5B)protein by cultured adipose derived stem cells.

FIG. 17 depicts detection of endothelial progenitor cells within adiposederived stem cell populations.

FIGS. 18A and 18B depict the in vitro development of vascular structuresin both normal (7A) and streptozotocin-treated (7B) mice.

FIG. 19 depicts the increased average restoration of blood flow inhindlimb ischemia mice treated with adipose derived stem cell comparedto a negative control.

FIGS. 20A and 20B shows that increasing adipose derived stem cell doseimproves graft survival and angiogenesis (20A) and depicts the retentionof adipose tissue architecture in histologic specimen (20B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for promoting wound healing usingadipose derived regenerative cells (“ADCs”). The present invention isbased, in part, on the discovery that the regenerative cells of theinvention (1) express angiogenic growth factors and cytokines, includingPIGF, VEGF, bFGF, IGF-II, Eotaxin, G-CSF, GM-CSF, IL-12 p40/p70, IL-12p70, IL-13, IL-6, IL-9, Leptin, MCP-1, M-CSF, MIG, PF-4, TIMP-1, TIMP-2,TNF-α, and Thrombopoetin, (2) secrete wound healing related cytokines,including MIP-1alpha, RANTES, MCP-1, MIG, TARC, MIP-1, KC and TIMP, (3)secrete collagen in vitro, and (4) promote wound healing in vivo.Accordingly, regenerative cells derived from adipose tissue, e.g., stemcells that express cell surface molecules generally considered to becharacteristic of mesenchymal stem cells have the capacity to promotenew vessel formation and wound healing.

The present invention also relates to rapid and reliable systems andmethods for separating and concentrating regenerative cells, e.g., stemcells and/or progenitor cells, from a wide variety of tissues, includingbut not limited to, adipose, bone marrow, blood, skin, muscle, liver,connective tissue, fascia, brain and other nervous system tissues, bloodvessels, and other soft or liquid tissues or tissue components or tissuemixtures (e.g., a mixture of tissues including skin, blood vessels,adipose, and connective tissue). In a preferred embodiment, the systemseparates and concentrates regenerative cells from adipose tissue. Inanother preferred embodiment, the system is automated such that theentire method may be performed with minimal user intervention orexpertise. In a particularly preferred embodiment, the regenerativecells obtained using the systems and methods of the present inventionare suitable for direct placement into a recipient suffering.

Preferably, the entire procedure from tissue extraction throughseparating, concentrating and placement of the regenerative cells intothe recipient would all be performed in the same facility, indeed, evenwithin the same room of the patient undergoing the procedure. Theregenerative cells may be used in a relatively short time period afterextraction and concentration. For example, the regenerative cells may beready for use in about one hour from the harvesting of tissue from apatient, and in certain situations, may be ready for use in about 10 to40 minutes from the harvesting of the tissue. In a preferred embodiment,the regenerative cells may be ready to use in about 20 minutes from theharvesting of tissue. The entire length of the procedure from extractionthrough separating and concentrating may vary depending on a number offactors, including patient profile, type of tissue being harvested andthe amount of regenerative cells required for a given therapeuticapplication. The cells may also be placed into the recipient incombination with other cells, tissue, tissue fragments, scaffolds orother stimulators of cell growth and/or differentiation in the contextof a single operative procedure with the intention of deriving atherapeutic, structural, or cosmetic benefit to the recipient. It isunderstood that any further manipulation of the regenerative cellsbeyond the separating and concentrating phase of the system will requireadditional time commensurate with the manner of such manipulation.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, “regenerative cells” refers to any heterogeneous orhomologous cells obtained using the systems and methods of the presentinvention which cause or contribute to complete or partial regeneration,restoration, or substitution of structure or function of an organ,tissue, or physiologic unit or system to thereby provide a therapeutic,structural or cosmetic benefit. Examples of regenerative cells include:ASCs, endothelial cells, endothelial precursor cells, endothelialprogenitor cells, macrophages, fibroblasts, pericytes, smooth musclecells, preadipocytes, differentiated or de-differentiated adipocytes,keratinocytes, unipotent and multipotent progenitor and precursor cells(and their progeny), and lymphocytes.

One mechanism by which the regenerative cells may provide a therapeutic,structural or cosmetic benefit is by incorporating themselves or theirprogeny into newly generated, existing or repaired tissues or tissuecomponents. For example, ASCs and/or their progeny may incorporate intonewly generated bone, muscle, or other structural or functional tissueand thereby cause or contribute to a therapeutic, structural or cosmeticimprovement. Similarly, endothelial cells or endothelial precursor orprogenitor cells and their progeny may incorporate into existing, newlygenerated, repaired, or expanded blood vessels to thereby cause orcontribute to a therapeutic, structural or cosmetic benefit.

Another mechanism by which the regenerative cells may provide atherapeutic, structural or cosmetic benefit is by expressing and/orsecreting molecules, e.g., growth factors, that promote creation,retention, restoration, and/or regeneration of structure or function ofa given tissue or tissue component. For example, regenerative cells mayexpress and/or secrete molecules which result in enhanced growth oftissues or cells that then participate directly or indirectly inimproved structure or function. Regenerative cells may express and/orsecrete growth factors, including, for example, Vascular EndothelialGrowth Factor (VEGF), Placental Growth factor (PIGF), bFGF, IGF-II,Eotaxin, G-CSF, GM-CSF, IL-12 p40/p70, EL-12 p70, IL-13, IL-6, IL-9,Leptin, MCP-1, M-CSF, MIG, PF-4, TIMP-1, TIMP-2, TNF-α, Thrombopoetin,and their isoforms, which may perform one or more of the followingfunctions: stimulate development of new blood vessels, i.e., promoteangiogenesis; improve oxygen supply of pre-existent small blood vessels(collaterals) by expanding their blood carrying capacity; inducemobilization of regenerative cells from sites distant from the site ofinjury to thereby enhance the homing and migration of such cells to thesite of injury; stimulate the growth and/or promote the survival ofcells within a site of injury thereby promoting retention of function orstructure; deliver molecules with anti-apoptotic properties therebyreducing the rate or likelihood of cell death and permanent loss offunction; and interact with endogenous regenerative cells and/or otherphysiological mechanisms.

The regenerative cells may be used in their ‘native’ form as present inor separated and concentrated from the tissue using the systems andmethods of the present invention or they may be modified by stimulationor priming with growth factors or other biologic response modifiers, bygene transfer (transient or stable transfer), by furthersub-fractionation of the resultant population on the basis or physicalproperties (for example size or density), differential adherence to asolid phase material, expression of cell surface or intracellularmolecules, cell culture or other ex vivo or in vivo manipulation,modification, or fractionation as further described herein. Theregenerative cells may also be used in combination with other cells ordevices such as synthetic or biologic scaffolds, materials or devicesthat deliver factors, drugs, chemicals or other agents that modify orenhance the relevant characteristics of the cells as further describedherein.

As used herein, “regenerative cell composition” refers to thecomposition of cells typically present in a volume of liquid after atissue, e.g., adipose tissue, is washed and at least partiallydisaggregated. For example, a regenerative cell composition of theinvention comprises multiple different types of regenerative cells,including ASCs, endothelial cells, endothelial precursor cells,endothelial progenitor cells, macrophages, fibroblasts, pericytes,smooth muscle cells, preadipocytes, differentiated or de-differentiatedadipocytes, keratinocytes, unipotent and multipotent progenitor andprecursor cells (and their progeny), and lymphocytes. The regenerativecell composition may also contain one or more contaminants, such ascollagen, which may be present in the tissue fragments, or residualcollagenase or other enzyme or agent employed in or resulting from thetissue disaggregation process described herein.

As used herein, “wound healing” is intended to include all disorderscharacterized by any disease, disorder, syndrome, anomaly, pathology, orabnormal condition of the skin and/or underlying connective tissue,e.g., skin wounds following surgery, skin abrasions caused my mechanicaltrauma, caustic agents or burns, cornea following cataract surgery orcorneal transplants, mucosal epithelium wounds following infection ordrug therapy (e.g., respiratory, gastrointestinal, genitourinary,mammary, oral cavity, ocular tissue, liver and kidney), diabetic wounds,skin wounds following grafting, and regrowth of blood vessels followingangioplasty. Treatment of a wound, disease or disorder is within thegambit of regenerative medicine.

As used herein, the term “ischemia” refers to any localized tissueischemia due to reduction of the inflow or outflow of blood. The term“ischemic wound” refers to the inability for wound contraction andre-epithelialization due to inadequate oxygen. Wound healing alsoincludes non-healing wounds. Certain medical and surgical situations areassociated with a high risk for developing non-healing wounds, e.g.,diabetes (type I and II), chronic peripheral vascular disease,rheumatoid arthritis, congestive heart failure, arterial or venousulcers, lymphedema, obesity, exogenous steroid administration, vesseldisease wounds, surgery wound breakdown, chemical wounds and woundsresulting from chemotherapeutic or other immuonosupressive regimens.

As used herein, the term “angiogenesis” refers to the process by whichnew blood vessels are generated from existing vasculature and tissue(Folkman, 1995). The phrase “repair or remodeling” refers to thereformation of existing vasculature. The alleviation of tissue ischemiais critically dependent upon angiogenesis. The spontaneous growth of newblood vessels provides collateral circulation in and around an ischemicarea, improves blood flow, and alleviates the symptoms caused by theischemia. Angiogenesis mediated diseases and disorders include acutemyocardial infarction, ischemic cardiomyopathy, peripheral vasculardisease, ischemic stroke, acute tubular necrosis, ischemicwounds—including AFT, sepsis, ischemic bowel disease, diabeticretinopathy, neuropathy and nephropathy, vasculitidies, ischemicencephalopathy, erectile dysfunction-physiologic, ischemic or traumaticspinal cord injuries, multiple organ system failure, ischemic gumdisease, and transplant related ischemia.

As used herein, “stem cell” refers to a multipotent regenerative cellwith the potential to differentiate into a variety of other cell types,which perform one or more specific functions and have the ability toself-renew. Some of the stem cells disclosed herein may be multipotent.

As used herein, “progenitor cell” refers to a multipotent regenerativecell with the potential to differentiate into more than one cell typeand has limited or no ability to self-renew. “Progenitor cell”, as usedherein, also refers to a unipotent cell with the potential todifferentiate into only a single cell type, which performs one or morespecific functions and has limited or no ability to self-renew. Inparticular, as used herein, “endothelial progenitor cell” refers to amultipotent or unipotent cell with the potential to differentiate intovascular endothelial cells.

As used herein, “precursor cell” refers to a unipotent regenerative cellwith the potential to differentiate into one cell type. Precursor cellsand their progeny may retain extensive proliferative capacity, e.g.,lymphocytes and endothelial cells, which can proliferate underappropriate conditions.

As used herein, the term “angiogenic factor” or “angiogenic protein”refers to any known protein, peptide or other agent capable of promotinggrowth of new blood vessels from existing vasculature (“angiogenesis”).Suitable angiogenic factors for use in the invention include, but arenot limited to, Placenta Growth Factor (Luttun et al., 2002), MacrophageColony Stimulating Factor (Aharinejad et al., 1995), GranulocyteMacrophage Colony Stimulating Factor (Buschmann et al., 2003), VascularEndothelial Growth Factor (VEGF)-A, VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGF-E (Mints et al., 2002), neuropilin (Wang et al., 2003), fibroblastgrowth factor (FGF)-1, FGF-2(bFGF), FGF-3, FGF4, FGF-5, FGF-6 (Botta etal., 2000), Angiopoietin 1, Angiopoietin 2 (Sundberg et al., 2002),erythropoietin (Ribatti et al., 2003), BMP-2, BMP4, BMP-7 (Carano andFilvaroff, 2003), TGF-beta (Xiong et al., 2002), IGF-1 (Shigematsu etal., 1999), Osteopontin (Asou et al., 2001), Pleiotropin (Beecken etal., 2000), Activin (Lamouille et al., 2002), Endothelin-1 (Bagnato andSpinella, 2003) and combinations thereof. Angiogenic factors can actindependently, or in combination with one another. When in combination,angiogenic factors can also act synergistically, whereby the combinedeffect of the factors is greater than the sum of the effects of theindividual factors taken separately. The term “angiogenic factor” or“angiogenic protein” also encompasses functional analogues of suchfactors. Functional analogues include, for example, functional portionsof the factors. Functional analogues also include anti-idiotypicantibodies which bind to the receptors of the factors and, thus, mimicthe activity of the factors in promoting angiogenesis and/or tissueremodeling. Methods for generating such anti-idiotypic antibodies arewell known in the art and are described, for example, in WO 97/23510,the contents of which are incorporated by reference herein.

Angiogenic factors used in the present invention can be produced orobtained from any suitable source. For example, the factors can bepurified from their native sources, or produced synthetically or byrecombinant expression. The factors can be administered to patients as aprotein composition. Alternatively, the factors can be administered inthe form of an expression plasmid encoding the factors. The constructionof suitable expression plasmids is well known in the art. Suitablevectors for constructing expression plasmids include, for example,adenoviral vectors, retroviral vectors, adeno-associated viral vectors,RNA vectors, liposomes, cationic lipids, lentiviral vectors andtransposons.

As used herein “stem cell number” or “stem cell frequency” refers to thenumber of colonies observed in a clonogenic assay in which adiposederived cells (ADC) are plated at low cell density (<10,000 cells/well)and grown in growth medium supporting MSC growth (for example, DMEM/F12medium supplemented with 10% fetal calf serum, 5% horse serum, andantibiotic/antimycotic agents). Cells are grown for two weeks afterwhich cultures are stained with hematoxylin and colonies of more than 50cells are counted as CFU-F. Stem cell frequency is calculated as thenumber of CFU-F observed per 100 nucleated cells plated (for example; 15colonies counted in a plate initiated with 1,000 nucleated regenerativecells gives a stem cell frequency of 1.5%). Stem cell number iscalculated as stem cell frequency multiplied by the total number ofnucleated ADC cells obtained. A high percentage (˜100%) of CFU-F grownfrom regenerative cells express the cell surface molecule CD105 which isalso expressed by marrow-derived stem cells (Barry et al., 1999). CD105is also expressed by adipose tissue-derived stem cells (Zuk et al.,2002).

As used herein, the term “adipose tissue” refers to fat including theconnective tissue that stores fat. Adipose tissue contains multipleregenerative cell types, including ASCs and endothelial progenitor andprecursor cells.

As used herein, the term “unit of adipose tissue” refers to a discreteor measurable amount of adipose tissue. A unit of adipose tissue may bemeasured by determining the weight and/or volume of the unit. Based onthe data identified above, a unit of processed lipoaspirate, as removedfrom a patient, has a cellular component in which at least 0.1% of thecellular component is stem cells; that is, it has a stem cell frequency,determined as described above, of at least 0.1%. In reference to thedisclosure herein, a unit of adipose tissue may refer to the entireamount of adipose tissue removed from a patient, or an amount that isless than the entire amount of adipose tissue removed from a patient.Thus, a unit of adipose tissue may be combined with another unit ofadipose tissue to form a unit of adipose tissue that has a weight orvolume that is the sum of the individual units.

As used herein, the term “portion” refers to an amount of a materialthat is less than a whole. A minor portion refers to an amount that isless than 50%, and a major portion refers to an amount greater than 50%.Thus, a unit of adipose tissue that is less than the entire amount ofadipose tissue removed from a patient is a portion of the removedadipose tissue.

As used herein, the term “processed lipoaspirate” refers to adiposetissue that has been processed to separate the active cellular component(e.g., the component containing regenerative) from the mature adipocytesand connective tissue. This fraction is referred to herein as“adipose-derived cells” or “ADC.” Typically, ADC refers to the pellet ofregenerative cells obtained by washing and separating and concentratingthe cells from the adipose tissue. The pellet is typically obtained bycentrifuging a suspension of cells so that the cells aggregate at thebottom of a centrifuge chamber or cell concentrator.

As used herein, the terms “administering,” “introducing,” “delivering,”“placement” and “transplanting” are used interchangeably herein andrefer to the placement of the regenerative cells of the invention into asubject by a method or route which results in at least partiallocalization of the regenerative cells at a desired site. Theregenerative cells can be administered by any appropriate route whichresults in delivery to a desired location in the subject where at leasta portion of the cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years.

As used herein, the term “treating” includes reducing or alleviating atleast one adverse effect or symptom of a disease or disorder As usedherein, “therapeutically effective dose of regenerative cells” refers toan amount of regenerative cells that are sufficient to bring about abeneficial or desired clinical effect. Said dose could be administeredin one or more administrations. However, the precise determination ofwhat would be considered an effective dose may be based on factorsindividual to each patient, including, but not limited to, the patient'sage, size, type or extent of disease, stage of the disease, route ofadministration of the regenerative cells, the type or extent ofsupplemental therapy used, ongoing disease process and type of treatmentdesired (e.g., aggressive vs. conventional treatment).

As used herein, the term “subject” includes warm-blooded animals,preferably mammals, including humans. In a preferred embodiment, thesubject is a primate. In an even more preferred embodiment, the subjectis a human.

As previously set forth herein, regenerative cells, e.g., stem andprogenitor cells, can be harvested from a wide variety of tissues. Thesystem of the present invention may be used for all such tissues.Adipose tissue, however, is an especially rich source of regenerativecells. Accordingly, the system of the present invention is illustratedherein using adipose tissue as a source of regenerative cells by way ofexample only and not limitation.

Adipose tissue can be obtained by any method known to a person ofordinary skill in the art. For example, adipose tissue may be removedfrom a patient by liposuction (syringe or power assisted) or bylipectomy, e.g., suction-assisted lipoplasty, ultrasound-assistedlipoplasty, and excisional lipectomy or combinations thereof. Theadipose tissue is removed and collected and may be processed inaccordance with any of the embodiments of a system of the inventiondescribed herein. The amount of tissue collected depends on numerousfactors, including the body mass index and age of the donor, the timeavailable for collection, the availability of accessible adipose tissueharvest sites, concomitant and pre-existing medications and conditions(such as anticoagulant therapy), and the clinical purpose for which thetissue is being collected. For example, the regenerative cell percentageof 100 ml of adipose tissue extracted from a lean individual is greaterthan that extracted from an obese donor (Table I). This likely reflectsa dilutive effect of the increased fat content in the obese individual.Therefore, it may be desirable, in accordance with one aspect of theinvention, to obtain larger amounts of tissue from overweight donorscompared to the amounts that would be withdrawn from leaner patients.This observation also indicates that the utility of this invention isnot limited to individuals with large amounts of adipose tissue.

TABLE I Effect of Body Mass Index on Tissue and Cell Yield Amount ofTissue Total Regenerative Cell Body Mass Index Status Obtained (g) Yield(×10⁷) Normal 641 ± 142 2.1 ± 0.4 Obese 1,225 ± 173   2.4 ± 0.5 p value0.03 0.6

After the adipose tissue is processed, the resulting regenerative cellsare substantially free from mature adipocytes and connective tissue.Accordingly, the system of the present invention generates aheterogeneous plurality of adipose derived regenerative cells which maybe used for research and/or therapeutic purposes. In a preferredembodiment, the cells are suitable for placement or re-infusion withinthe body of a recipient. In other embodiments, the cells may be used forresearch, e.g., the cells can be used to establish stem or progenitorcell lines which can survive for extended periods of time and be usedfor further study.

Reference will now be made in detail to the presently preferredembodiments of the invention, examples of which are illustrated in theaccompanying drawings. Wherever possible, the same or similar referencenumbers are used in the drawings and the description to refer to thesame or like parts. It should be noted that the drawings are insimplified form and are not to precise scale. In reference to thedisclosure herein, for purposes of convenience and clarity only,directional terms, such as, top, bottom, left, right, up, down, over,above, below, beneath, rear, front, distal, and proximal are used withrespect to the accompanying drawings. Such directional terms should notbe construed to limit the scope of the invention in any manner.

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation. The intent of thefollowing detailed description, although discussing exemplaryembodiments, is to be construed to cover all modifications,alternatives, and equivalents of the embodiments as may fall within thespirit and scope of the invention as defined by the appended claims. Thepresent invention may be utilized in conjunction with various medicalprocedures that are conventionally used in the art.

Referring now to the Figures, a system 10 of the present invention isgenerally comprised of one or more of a tissue collection chamber 20, aprocessing chamber 30, a waste chamber 40, an output chamber 50 and asample chamber 60. The various chambers are coupled together via one ormore conduits 12 such that fluids containing biological material maypass from one chamber to another while maintaining a closed, sterilefluid/tissue pathway. The conduits may comprise rigid or flexible bodiesreferred to interchangeably herein as lumens and tubing, respectively.In certain embodiments, the conduits are in the form of flexible tubing,such as polyethylene tubing conventionally used in clinical settings,silicone or any other material known in the art. The conduits 12 canvary in size depending on whether passage of fluid or tissue is desired.The conduits 12 may also vary in size depending on the amount of tissueor fluid that is cycled through the system. For example, for the passageof fluid, the conduits may have a diameter ranging from about 0.060 toabout 0.750 inches and for the passage of tissue, the conduits may havea diameter ranging from 0.312 to 0.750 inches. Generally, the size ofthe conduits is selected to balance the volume the conduits canaccommodate and the time required to transport the tissue or fluidsthrough said conduits. In automated embodiments of the system, theforegoing parameters, i.e., volume and time for transport, must beidentified such that the appropriate signals can be transmitted to theprocessing device of the system. This allows the device to move accuratevolumes of liquid and tissue from one chamber to another. The flexiletubing used should be capable of withstanding negative pressure toreduce the likelihood of collapse. The flexible tubing used should alsobe capable of withstanding positive pressure which is generated by, forexample, a positive displacement pump, which may be used in the system.

All the chambers of the system may be comprised of one or more ports,e.g., outlet 22 or inlet 21 ports, which accept standard IV, syringe andsuction tubing connections. The ports may be a sealed port such as arubber septum closed syringe needle access port 51. The inlet ports maybe coupled to one or more cannulas (not shown) by way of conduits. Forexample, a tissue inlet port 21 may be coupled to an integrated singleuse liposuction cannula and the conduit may be a flexible tubing. Theconduits are generally positioned to provide fluid passageways from onechamber of the system to another. Towards this end, the conduits andports may be coupled to, for example, a suction device (not shown) whichmay be manually or automatically operated. The suction device may be,e.g., a syringe or an electric pump. The suction device should becapable of providing sufficient negative pressure to aspirate tissuefrom a patient. Generally, any suitable suction device known to one ofordinary skill in the art, e.g., a surgeon, may be used.

The conduits 12 may further comprise one or more clamps (not shown) tocontrol the flow of material among various components of the system. Theclamps are useful for maintaining the sterility of the system byeffectively sealing different regions of the system. Alternatively, theconduits 12 may comprise one or more valves 14 that control the flow ofmaterial through the system. The valves 14 are identified as opencircles in the Figures. In preferred embodiments, the valves may beelectromechanical pinch valves. In another embodiment, the valves may bepneumatic valves. In yet other embodiments, the valves may be hydraulicvalves or mechanical valves. Such valves are preferably activated by acontrol system which may be coupled to levers. The levers may bemanually manipulated such that the levers are activated. In automatedembodiments, the control system may be coupled to the levers as well asto a processing device which may activate the valves at pre-determinedactivation conditions. In certain automated embodiments, activation ofthe valves may be partially automated and partially subject to theuser's preference such that the process may be optimized. In yet otherembodiments, certain valves may be activated manually and othersautomatically through the processing device. The valves 14 may also beused in conjunction with one or more pumps, e.g., peristaltic pumps 34or positive displacement pumps (not shown). The conduits 12 and/or thevalves 14 may also be comprised of sensors 29, e.g., optical sensors,ultrasonic sensors, pressure sensors or other forms of monitors known inthe art that are capable of distinguishing among the various fluidcomponents and fluid levels that flow through the system. In a preferredembodiment, the sensors 29 may be optical sensors.

The system may also include a plurality of filters 36. In certainembodiments, the filters may be within a chamber of the system 28.Different chambers within the system may be comprised of differentfilters. The filters are effective to separate the regenerative cells,e.g., stem cells and/or progenitor cells, from undesirable cells anddisaggregation agents that may be used in accordance with the system. Inone embodiment, a filter assembly 36 includes a hollow fiber filtrationdevice. In another embodiment, a filter assembly 36 includes apercolative filtration device, which may or may not be used with asedimentation process. In a further embodiment, the filter assembly 36comprises a centrifugation device, which may or may not be used with anelutriation device and process. In yet another embodiment, the systemcomprises a combination of these filtering devices. The filtrationfunctions of the present invention can be two-fold, with some filtersremoving things from the final concentration such as collagen, freelipid, free adipocytes and residual collagenase, and with other filtersbeing used to concentrate the final product. The filters of the systemmay be comprised of a plurality of pores ranging in diameters and/orlength from 20 to 800 μm. In a preferred embodiment, the collectionchamber 20 has a prefixed filter 28 with a plurality of pores rangingfrom 80 to 400 μm. In another preferred embodiment, the collectionchamber 20 has a prefixed filter 28 with a plurality of 265 μm pores. Inother embodiments, the filters may be detachable and/or disposable.

The system may also be comprised of one or more temperature controldevices (not shown) that are positioned to adjust the temperature of thematerial contained within one or more chambers of the system. Thetemperature control device may be a heater, a cooler or both, i.e., itmay be able to switch between a heater and a cooler. The temperaturedevice may adjust the temperature of any of the material passing throughthe system, including the tissue, the disaggregation agents, theresuspension agents, the rinsing agents, the washing agents or theadditive's. For example, heating of adipose tissue facilitatesdisaggregation whereas the cooling of the regenerative cell output isdesirable to maintain viability. Also, if pre-warmed reagents are neededfor optimal tissue processing, the role of the temperature device wouldbe to maintain the pre-determined temperature rather than to increase ordecrease the temperature.

To maintain a closed, sterile fluid/tissue pathway, all ports and valvesmay comprise a closure that maintains the sealed configuration of thesystem. The closure may be a membrane that is impermeable to fluid, airand other contaminants or it may be any other suitable closure known inthe art. Furthermore, all ports of the system may be designed such thatthey can accommodate syringes, needles or other devices for withdrawingthe materials in the chambers without compromising the sterility of thesystem.

As set forth herein, tissue may be extracted from a patient via any artrecognized method. The aspirated tissue may be extracted prior to beingplaced in the system for processing. The aspirated tissue is typicallytransferred to the collection chamber 20 through conduits 12 via asealed entry port, such as a rubber septum closed syringe needle accessport (not shown on collection chamber). Alternatively, the tissueextraction step may be part of the system. For example, the collectionchamber 20 may be comprised of a vacuum line 11 which facilitates tissueremoval using a standard cannula inserted into the patient. Thus, inthis embodiment, the entire system is attached to the patient. Thetissue may be introduced into the collection chamber 20 through an inletport 21 via a conduit such as 12 a which are part of a closed sterilepathway. The collection chamber 20 may be comprised of a plurality offlexible or rigid canisters or cylinders or combinations thereof. Forexample, the collection chamber 20 may be comprised of one or more rigidcanisters of varying sizes. The collection chamber 20 may also becomprised of one or more flexible bags. In such systems, the bag ispreferably provided with a support, such as in internal or externalframe, that helps reduce the likelihood that the bag will collapse uponthe application of suction to the bag. The collection chamber 20 issized to hold the requisite amount of saline to appropriately wash anddisaggregate the tissue prior to the wash and concentrate stage of theprocess performed in the processing chamber 30. Preferably, the volumeof tissue or fluid present in the collection chamber 20 is easilyascertainable to the naked eye. For example, to obtain regenerativecells from adipose tissue, a suitable collection chamber has thecapacity to hold 800 ml of lipoaspirate and 1200 ml of saline.Accordingly, in one embodiment, the collection chamber 20 has a capacityof at least 2 liters. In another embodiment, to separate and concentratered blood cells from blood, the collection chamber 20 has a capacity ofat least 1.5 liters. Generally, the size of the collection chamber 20will vary depending on the type and amount of tissue collected from thepatient. The collection chamber 20 may be sized to hold as little asabout 5 ml to up to about 2 liters of tissue. For smaller tissuevolumes, e.g., 5 mls to 100 mls, the tissue may be gathered in a syringeprior to transfer to the collection chamber 20.

The collection chamber 20 may be constructed using any suitablebiocompatible material that can be sterilized. In a preferredembodiment, the collection chamber 20 is constructed of disposablematerial that meets biocompatibility requirements for intravascularcontact as described in the ISO 10993 standard. For example,polycarbonate acrylic or ABS may be used. The fluid path of thecollection chamber 20 is preferably pyrogen free, i.e., suitable forblood use without danger of disease transmittal. In one embodiment, thecollection chamber 20 is constructed of a material that allows the userto visually determine the approximate volume of tissue present in thechamber. In other embodiments, the volume of tissue and/or fluid in thecollection chamber 20 is determined by automated sensors 29. Thecollection chamber 20 is preferably designed such that in an automatedembodiment, the system can determine the volume of tissue and/or fluidwithin the chamber with a reasonable degree of accuracy. In a preferredembodiment, the system senses the volume within the collection chamberwith an accuracy of plus or minus fifteen percent.

In a particular embodiment provided by way of example only, thecollection chamber 20 is in the form of a rigid chamber, for example, achamber constructed of a medical grade polycarbonate containing aroughly conical prefixed filter 28 of medical grade polyester with amesh size of 265 μm (see FIG. 5). The rigid tissue collection containermay have a size of approximately eight inches high and approximatelyfive inches in diameter; the wall thickness may be about 0.125 inches.The interior of the cylinder may be accessed through, for example, oneor more ports for suction tubing, one or more ports with tubing forconnection through sterile docking technology, and/or one or more portsfor needle puncture access through a rubber septum. The prefixed filter28 in the interior of the collection chamber 20 is preferably structuredto retain adipose tissue and to pass non-adipose tissue as, for example,the tissues are removed from the patient. More specifically, the filter28 may allow passage of free lipid, blood, and saline, while retainingfragments of adipose tissue during, or in another embodiment after, theinitial harvesting of the adipose tissue. In that regard, the filter 28includes a plurality of pores, of either the same or different sizes,but ranging in size from about 20 μm to 5 mm. In a preferred embodiment,the filter 28 includes a plurality of 400 μm pores. In a preferredembodiment, the filter 28 is a medical grade polyester mesh of around200 μm thickness with a pore size of around 265 μm and around 47% openarea. This material holds the tissue during rinsing but allows cells topass out through the mesh following tissue disaggregation. Thus, whenthe tissues are aspirated from the patient, non-adipose tissue may beseparated from adipose tissue. The same functionality could be achievedwith different materials, mesh size, and the number and type of ports.For example, mesh pore sizes smaller than 100 μm or as large as severalthousand microns would achieve the same purpose of allowing passage ofsaline and blood cells while retaining adipose tissue aggregates andfragments. Similarly, the same purpose could be achieved by use of analternative rigid plastic material, or by many other modifications thatwould be known to those skilled in the art

The system 10 may also be comprised of one or more solution sources 22.The solution source may comprise a washing solution source 23, and atissue disaggregation agent source 24, such as collagenase. Thecollection chamber 20 is comprised of closed fluid pathways that allowsfor the washing and disaggregating solutions or agents to be added tothe tissue in an aseptic manner.

The containers for the washing solution 23 and the disaggregation agents24 may be any suitable container that can hold their contents in asterile manner, e.g., a collapsible bag, such as an IV bag used inclinical settings. These containers may have conduits 12, such asconduit 12 e, coupled to the collection chamber 20 so that the washingsolution and the disaggregation agent may be delivered to the interiorof the collection chamber 20. The washing solution and thedisaggregation agent may be delivered to the interior of the collectionchamber 20 through any art-recognized manner, including simple gravitypressure applied to the outside of the containers for the saline 23and/or the disaggregation agents 24 or by placement of a positivedisplacement pump on the conduits, e.g., conduit 12 d in FIG. 4. Inautomated embodiments, the processing device of the system calculatesvarious parameters, e.g., the volume of saline and time or number ofcycles required for washing as well as the concentration or amount ofdisaggregation agent and the time required for disaggregation based oninformation initially entered by the user (e.g., volume of tissue beingprocessed). Alternatively, the amounts, times etc. can be manuallymanipulated by the user.

The tissue and/or fluid within the collection chamber should bemaintained at a temperature ranging from 30 degrees Celsius to 40degrees Celsius. In a preferred embodiment, the temperature of thesuspension inside the collection chamber is maintained at 37 degreesCelsius. In certain embodiments, if the surgical procedure ortherapeutic application needs to be delayed, the selected tissue may bestored in the collection chamber for later use. The tissue may be storedat or about room temperature or at about 4 degrees Celsius for up to 96hours.

The washing solution may be any solution known to one of skill in theart, including saline or any other buffered or unbuffered electrolytesolution. The types of tissue being processed will dictate the types orcombinations of washing solutions used. Typically, the washing solution,such as saline, enters the collection chamber 20 after the adiposetissue has been removed from the patient and placed in the collectionchamber. However, the washing solution may be delivered to thecollection chamber 20 before the adipose tissue is extracted, or may bedelivered to the collection chamber 20 concurrently with the adiposetissue. In the collection chamber 20, the washing solution and theextracted adipose tissue may be mixed by any means including the methodsdescribed below.

For example, the tissue may be washed by agitation (which maximizes cellviability and minimizes the amount of free lipid released). In oneembodiment, the tissue is agitated by rotating the entire collectionchamber 20 through an arc of varying degrees (e.g., through an arc ofabout 45 degrees to about 90 degrees) at varying speeds, e.g., about 30revolutions per minute. In other embodiments, the tissue is agitated byrotating the entire collection chamber 20, wherein the collectionchamber 20 is comprised of one or more paddles or protrusions rigidlyattached to an inside surface of the collection chamber, through an arcof varying degrees (e.g., through an arc of about 45 degrees to about 90degrees) at varying speeds, e.g., about 30 revolutions per minute. Therotation of the collection chamber 20 described above may beaccomplished by a drive mechanism attached to or in proximity with thecollection chamber 20. The drive mechanism may be a simple belt or gearor other drive mechanism known in the art. The speed of the rotation maybe, for example, 30 revolutions per minute. Generally, higher speedshave been found to generate larger volumes of free lipids and may not beoptimal.

In other embodiments, the tissue is agitated by placing a rotatableshaft 25 inside the collection chamber 20, wherein the rotatable shaftis comprised of one or more paddles 25 a or protrusions rigidly attachedto the rotatable shaft 25 which pass through the mixture as the shaft isbeing rotated. In certain embodiments, the rotatable shaft 25 withrigidly attached 25 a paddles may be rested on the bottom of thecollection chamber 20. This may be accomplished, for example, by placingthe paddle-like device into a spinning magnetic field (e.g., magneticstirrer). Alternatively, agitating of the tissue may be accomplishedusing a simple agitator known in the art, i.e. a device implementingshaking up and down without rotation. The tissue may also be washedusing any other art-recognized means including rocking, stirring,inversion, etc.

After a desired amount of wash cycles, a tissue disaggregation agent maybe delivered to the collection chamber 20 to separate the regenerativecells from the remaining adipose tissue components. The disaggregationagent may be any disaggregation agent known to one of skill in the art.Disaggregation agents that may be used include neutral proteases,collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, membersof the Blendzyme enzyme mixture family, e.g., Liberase H1, pepsin,ultrasonic or other physical energy, lasers, microwaves, othermechanical devices and/or combinations thereof. A preferreddisaggregation agent of the invention is collagenase. The disaggregationagents may be added with other solutions. For example, saline, such assaline delivered from a saline source 23 as described above, may beadded to the adipose tissue along with or immediately followed byaddition of collagenase. In one embodiment, the washed adipose tissue ismixed with a collagenase-containing enzyme solution at or around 37° C.for about 20-60 minutes. In other embodiments, a higher concentration ofcollagenase or similar agent may be added to decrease the digestiontime. The washed adipose tissue and the tissue disaggregation agent maythen be agitated in manners similar to the agitation methods describedabove, until the washed adipose tissue is disaggregated. For example,the washed adipose tissue and the tissue disaggregation agent may beagitated by rotating the entire collection chamber through an arc ofapproximately 90 degrees, by having a shaft which contains one or morepaddles which pass through the solution as the shaft is being rotated,and/or by rotating the entire collection chamber which contains paddlesor protrusions on the inside surface of the collection chamber.

Depending on the purpose for which the adipose derived cells will beused, the adipose tissue may either be partially disaggregated, orcompletely disaggregated. For example, in embodiments in which theadipose derived cells are to be combined with a unit of adipose tissue,it may be desirable to partially disaggregate the harvested adiposetissue, to remove a portion of the partially disaggregated adiposetissue, and then continue disaggregating the remaining portion ofadipose tissue remaining in the collection chamber. Alternatively, aportion of washed adipose tissue may be removed and set aside in asample container prior to any digestion. In another embodiment,harvested adipose tissue is partially disaggregated to concentrate cellsbefore being reintroduced back into the patient. In one embodiment, theadipose tissue is mixed with a tissue disaggregation agent for a periodof time generally less than about 20 minutes. A portion of the partiallydisaggregated tissue may then be removed from the collection chamber,and the remaining partially disaggregated tissue may be furtherdisaggregated by mixing the adipose tissue with a tissue disaggregationagent for another 40 minutes. When the adipose derived cells are to beused as an essentially pure population of regenerative cells, theadipose tissue may be fully disaggregated.

After digestion, the tissue and disaggregation agent solution is allowedto settle for a period of time sufficient to allow the buoyant andnon-buoyant components of the solution to differentiate within thecollection chamber. Typically, the time ranges from about 15 seconds toseveral minutes but other times may be implemented in modifiedembodiments. The buoyant layer is comprised of the regenerative cellsthat require further washing and concentrating. The non-buoyant layercomprises blood, collagen, lipids and other non-regenerative cellcomponents of the tissue. The non-buoyant layer must be removed to thewaste chamber.

Accordingly, the collection chamber 20 is preferably comprised of anoutlet port 22 at the lowest point of the chamber such that blood andother non-buoyant components of the tissue may be drained to one or morewaste containers 40 via one or more conduits 12. The collection chamber20 is generally in (or may be placed in) an upright position such thatthe outlet ports 22 are located at the bottom of the collection chamber.The draining may be passive or active. For example, the non-buoyantcomponents described above could be drained using gravity, by applyingpositive or negative pressure, by use of pumps 34 or by use of vents 32.In automated embodiments, the processing device can signal certainvalves and/or pumps to drain the non-buoyant layer from the collectionchamber 20. The automated embodiments may also be comprised of sensors29 which can detect when the interface between the buoyant andnon-buoyant liquids has been reached. The automated embodiments may alsobe comprised of a sensor 29, e.g., an optical sensor, which may becapable of detecting a change in the light refraction of the effluentwhich is flowing in the conduit leading out of the collection chamber.The appropriate change in the light refraction may signal the presenceof the buoyant layer in the outgoing conduits which indicates that thenon-buoyant layer has been drained. The sensor 29 can then signal theprocessing device to proceed with the next step.

In certain embodiments however, the tissue may be processed to retrievethe non-regenerative cell component of the tissue. For example, incertain therapeutic or research applications, collagen, proteins, matrixor stromal components, lipids, adipocytes or other components of thetissue may be desired. In such embodiments, it is the buoyant layercomprising the regenerative cells that must be removed as describedabove to the waste chamber. The non-buoyant layer is then retained inthe system for further processing as needed.

Once the non-buoyant layer is removed, the buoyant layer comprising theregenerative cells may be washed one or more times to remove residualcontaminants. Accordingly, the collection chamber 20 typically includesone or more ports 21 for permitting the washing solution to be deliveredto the interior of the chamber, and one or more ports 22 for permittingwaste and other materials to be directed out from the collection chamber20. For example, the collection chamber may include one or more sealedentry ports as described herein. The collection chamber 20 may alsoinclude one or more caps (not shown), such as a top cap and a bottom capto further ensure that the system remains sterile while washing solutionis delivered into the collection chamber and/or waste is transportedout. The ports 21 may be provided on the caps of the collection chamberor on a sidewall of the collection chamber.

The process of washing with fresh wash solution may be repeated untilthe residual content of non-buoyant contaminants in the solution reachesa pre-determined level. In other words, the remaining material in thecollection chamber 20, which comprises the buoyant material of themixture described above, including adipose tissue fragments, may bewashed one or more additional times until the amount of undesiredmaterial is reduced to a desired pre-determined level. One method ofdetermining the end point of the washing is to measure the amount of redblood cells in the tissue solution. This can be accomplished bymeasuring the light absorbed on the 540 nm wavelength. In a preferredembodiment, a range between about 0.546 and about 0.842 is deemedacceptable.

During the washing and/or disaggregation, one or more additives may beadded to the various containers as needed to enhance the results. Someexamples of additives include agents that optimize washing anddisaggregation, additives that enhance the viability of the active cellpopulation during processing, anti-microbial agents (e.g., antibiotics),additives that lyse adipocytes and/or red blood cells, or additives thatenrich for cell populations of interest (by differential adherence tosolid phase moieties or to otherwise promote the substantial reductionor enrichment of cell populations). Other possible additives includethose that promote recovery and viability of regenerative cells (forexample, caspase inhibitors) or which reduce the likelihood of adversereaction on infusion or emplacement (for example, inhibitors ofre-aggregation of cells or connective tissue).

After a sufficient settling time has elapsed, the non-buoyant fractionof the resulting mixture of washed adipose tissue fragments and tissuedisaggregation agents will contain regenerative cells, e.g., stem cellsand other adipose derived progenitor cells. As discussed herein, thenon-buoyant fraction containing the regenerative cells will betransferred to the processing chamber 30 wherein the regenerative cellsof interest, such as the adipose derived stem cells, will be separatedfrom other cells and materials present in the non-buoyant fraction ofthe mixture. This non-buoyant fraction is referred to herein as theregenerative cell composition and comprises multiple different types ofcells, including stem cells, progenitor cells, endothelial precursorcells, adipocytes and other regenerative cells described herein. Theregenerative cell composition may also contain one or more contaminants,such as collagen and other connective tissue proteins and fragmentsthereof, which were present in the adipose tissue fragments, or residualcollagenase from the tissue disaggregation process.

The processing chamber 30 of the invention is preferably positionedwithin the system such that the regenerative cell composition moves fromthe collection chamber 20 to the processing chamber 30 by way of tubing12, valves 14 and pump 34 in a sterile manner. The processing chamber issized to accommodate tissue/fluid mixtures ranging from 10 mL to 1.2 L.In a preferred embodiment, the processing chamber is sized toaccommodate 800 mLs. In certain embodiments, the entire regenerativecell composition from the collection chamber 20 is directed to theprocessing chamber 30. However, in other embodiments, a portion of theregenerative cell composition is directed to the processing chamber 30,and another portion is directed to a different region of the system,e.g., the sample chamber 60, to be recombined with cells processed inthe processing chamber 30 at a later time.

The processing chamber 30 may be constructed using any suitablebiocompatible material that can be sterilized. In a preferredembodiment, the processing chamber 30 is constructed of disposablematerial that meets biocompatibility requirements for intravascularcontact, as described in the ISO 10993 standard. For example,polycarbonate, acrylic, ABS, ethylene vinyl acetate or styrene-butadienecopolymers (SBC) may be used. In another embodiment, the fluid path ofthe disposable processing chamber is pyrogen free. The processingchamber may be in the form of a plastic bag, such as thoseconventionally used in processing blood in blood banks; or in otherembodiments, it may be structurally rigid (FIG. 6). In one embodiment,the processing chamber 30 may be similar to the processing chamberdisclosed in commonly owned U.S. application Ser. No. 10/316,127, filedDec. 7, 2001 and U.S. application Ser. No. 10/325,728, filed Dec. 20,2002, the contents of which in their entirety are hereby incorporated byreference.

The processing chamber 30 may be constructed in any manner suitable forseparating and concentrating cells, including filtration andcentrifugation and/or combinations thereof. In certain embodiments, theregenerative cell composition from the collection chamber 20 isintroduced into the processing chamber 30 where the composition can befiltered to separate and/or concentrate a particular regenerative cellpopulation. Cell filtration is a method of separating particularcomponents and cells from other different components or types of cells.For example, the regenerative cell composition of the inventioncomprises multiple different types of cells, including stem cells,progenitor cells and adipocytes, as well as one or more contaminants,such as collagen, which was present in the adipose tissue fragments, orresidual collagenase from the tissue disaggregation process. The filters36 present in the processing chamber 30 may allow for separation andconcentration of a particular subpopulation of regenerative cells, e.g.,stem cells or endothelial progenitors cells etc.

Some variables which are associated with filtration of cells from aliquid include, but are not limited to, pore size of the filter media,geometry (shape) of the pore, surface area of the filter, flow directionof the solution being filtered, trans-membrane pressure, dilution of theparticular cell population, particulate size and shape as well as cellsize and cell viability. In accordance with the disclosure herein, theparticular cells that are desired to be separated or filtered aretypically adipose derived stem cells. However, in certain embodiments,the particular cells may include adipose derived progenitor cells, suchas endothelial precursor cells, alone or in combination with the stemcells.

The regenerative cell composition may be directed through a filterassembly, such as filter assembly 36. In certain embodiments, the filterassembly 36 comprises a plurality of filters which are structured toperform different functions and separate the regenerative cellcomposition into distinct parts or components. For example, one of thefilters may be configured to separate collagen from the regenerativecell composition, one of the filters may be configured to separateadipocytes and/or lipid components from the regenerative cellcomposition, and one of the filters may be configured to separateresidual enzymes, such as the tissue disaggregation agent, from theregenerative cell composition. In certain embodiments, one of thefilters is capable of performing two functions, such as separatingcollagen and the tissue disaggregation agent from the composition. Theplurality of filters are typically serially arranged; however, at leasta portion of the filters may be arranged in parallel, as well. A serialarrangement of the filters of the filter assembly 36 is shown in FIG. 2.A parallel arrangement of the filters of the filter assembly 36 is shownin FIG. 3.

In one embodiment, the filter assembly 36 comprises a first filter, asecond filter, and a third filter. The first filter is configured toremove collagen particles present in the regenerative cell composition.These collagen particles are typically approximately 0.1 microns indiameter and can be up to 20 microns long. The collagen particles may beof varying sizes depending on the digestion. They also may be fibrils,meaning they have twists and turns. Any of the filters described hereinmay be made from polyethersulfone, polyester, PTFE, polypropylene, PVDF,or possibly cellulose. There are two possibilities for filtering thecollagen. One is to try to remove the larger particles first, lettingthe cells go through, which would require for example a filter probablyin the 10 micron range. The second method is to use a smaller sizefilter, such as 4.5 micron, with the intent that the collagen would bewell digested, so as to trap the cells, and let the collagen passthrough. This would require a means to float the cells back off thefilter. There may also be a possibility of implementing a filter whichwould attract and hold the collagen fibers.

The second filter is configured to remove free immature adipocytes whichare not buoyant in the regenerative cell composition. In one embodimentthe second filter can be constructed of polyester and have a pore sizebetween about 30 and about 50 microns with a preferred pore size beingabout 40 microns. Although referred to as a second filter, placement ofsuch a device may be in a first, rather than second, position tofacilitate an initial removal of larger cells and particles. The thirdfilter is configured to remove the unused or residual collagenase orother tissue disaggregation agent present in the composition. In apreferred implementation, the collagenase may degenerate over time. Inone embodiment, the third filter comprises a plurality of pores having adiameter, or length less than 1 μm. In certain embodiments, the poresmay have diameters that are smaller than 1 μm. In other embodiments, thepores have diameters between 10 kD and 5 microns. In certainembodiments, the third filter may be configured to concentrate theregenerative cell population into a small volume of saline or otherwashing solution, as discussed herein. As presently preferred, only thefinal filter is the hollow fiber unit. It is not necessary for any ofthe filters to be of the hollow fiber type. The hollow fiber unit isused for the final filter in a preferred implementation because it isthe most efficient in removing the collagenase with the smallestdetrimental effect to the regenerative cells. In an embodiment whereinthe device is a collection of off the shelf items, the three filters arein separate housings. It is feasible to have the first and secondfilters combined into one housing if a hollow fiber unit is used for thethird filter. If the final filter is not a hollow fiber set-up then allthree filters can be contained in one housing.

The filters of the filter assembly 36 may be located in the processingchamber 30 or may be provided as components separate from the processingchamber 30. In addition, the filters of the filter assembly 36 may beprovided in multiple processing chambers or in an inline fashion. Incertain embodiments, the conduits or tubing may act as a processingchamber or chambers. The processing chamber can be reduced in size suchthat it becomes the inside volume of the conduits which connect thefilters. This type of system will function correctly if the volume oftissue solution is sized appropriately. Thus, the conduits may act asthe processing chamber by containing the fluid with cells as it is beingrun through the filters. Care may be taken to minimize the volume of theconduits so that cells/tissue are not unnecessarily lost in the processof priming and running the system.

Referring to the embodiment described above, the regenerative cellcomposition, containing the washed cells and residual collagen,adipocytes, and/or undigested tissue disaggregation agent, may bedirected through the first filter to remove at least a portion of andpreferably substantially all of the collagen particles from thecomposition so that fewer, and preferably no, collagen particles arepresent in the filtered solution. The filtered regenerative cellcomposition containing the adipocytes and/or undigested tissuedisaggregation agent, may then be directed through the second filter toremove at least a portion of and preferably substantially all of thefree adipocytes from the filtered regenerative cell composition.Subsequently, the twice filtered regenerative cell composition,containing the undigested tissue disaggregation agent, may be directedthrough the third filter, such as a hollow fiber filtration device, asdiscussed herein, to remove or reduce the undigested tissuedisaggregation agent from the regenerative cell composition.

The thrice-filtered regenerative cell composition (i.e., the compositionremaining after being passed through the first, second, and thirdfilters) may then be directed to multiple outlets, which may include aportion of the processing chamber 30 comprising multiple outlets. Theseoutlets can serve to maintain the necessary pressure, as well as toprovide connections via conduits to other containers which may includethe collection chamber 20, the output chamber 50, and/or the wastecontainer 40.

In one embodiment, a filter of the filter assembly 36 comprises ahollow-fiber filtration member. Or, in other words, the filter comprisesa collection of hollow tubes formed with the filter media. Examples offilter media which can be used with the disclosed system 10 includepolysulfone, polyethersulfone or a mixed ester material, and the like.These hollow fibers or hollow tubes of filter media may be contained ina cylindrical cartridge of the filter assembly 36. The individual tubesor fibers of filter media typically have an inside diameter which rangesfrom about 0.1 mm to about 1 mm with a preferred value being about 0.5mm. The diameter and length of a suitable cylindrical cartridge willdetermine the number of individual tubes of filter media which can beplaced inside the cartridge. One example of a suitable hollow fiberfilter cartridge is the FiberFlo® Tangential Flow Filter, catalog#M-C-050-K (Minntech, Minneapolis, Minn.). Pore sizes of the filtermedia can range between about 10 kiloDaltons and about 5 microns with apreferred pore size being about 0.5 microns.

In the hollow-fiber filter, each hollow tube has a body with a firstend, a second end, and a lumen located in the body and extending betweenthe first end and second end. The body of each hollow tube includes aplurality of pores. The pores are generally oriented in the body so thata regenerative cell composition is filtered by flowing through the lumenof the body, and the products to be filtered tangentially pass throughthe pores, as shown in FIG. 12A. In other words, the smaller particlesin the liquid pass tangentially through the pores relative the flow offluid through the lumen of the body. The composition with theregenerative cells passes through the lumen of each hollow tube when thecomposition is being filtered. Preferably, the flow of the compositionis tangential to the pores of the body of each hollow tube.

By using a tangential flow of fluid, the efficiency of filtration of thestem cells may be enhanced relative to other filtration techniques. Forexample, in accordance with some filtration techniques, the pores of thefilter media are placed in such a manner that the filter is orientatedperpendicular to the flow of the fluid so that the Filter media blocksthe path of the fluid being filtered, as illustrated in FIG. 12B. Inthis type of filtration, the particles which are being filtered out ofthe regenerative cell composition, e.g., the stem cells, tend to buildup on one side of the filter and block the flow of the fluid through thepores. This blockage can reduce the efficiency of the filter. Inaddition, the cells are constantly compressed by the pressure of thefluid flow as well as the weight of the cells accumulating on theupstream side of the filter. This can lead to increased lysis of stemcells. Thus, in such filtration techniques wherein the flow of fluid isparallel to the orientation of the pores in the filter, both large cellsand small particles can be undesirably directed against the filter mediaas the fluid is passed through the pores. Consequently, larger productsin the liquid such as cells may block the pores, thereby decreasing thefiltering effect and increasing an occurrence of cell rupture or injury.

In contrast, in the hollow fiber configuration of the present system 10,the fluid which is being filtered flows inside the lumen of the hollowtube. The portion of the fluid which has the ability to pass through thepores of the body of the filter does so with the aid of the positivepressure of the fluid on the inside of the body as well as a negativepressure which is applied on the outside of the body. In thisembodiment, the cells typically are not subjected to the pressure of thefluid flow or the weight of other cells, and therefore, the shear forceson the stem cells are reduced Thus, the efficiency and effectiveness ofthe filtration can be enhanced by the reduction in clogging rates andthe reduction in regenerative cell lysis. Due to the size of the salineand unwanted protein molecules, during filtration, these molecules andother small components pass through the pores of the bodies of thehollow tubes to the outside of the hollow tubes and are directed to thewaste container 40. In one embodiment, filtration is enhanced bygenerating a vacuum on the outside of the hollow tube filter media. Dueto the size of the regenerative cells, e.g., stem cells or progenitorcells, these cells typically cannot pass through the pores of the bodyand therefore remain on the inside of the hollow tube filter (e.g., inthe lumens of the tubes) and are directed back to the processing chamber30 via a conduit between the filter and the processing chamber, or tothe output chamber 50.

In one specific embodiment, the hollow fiber filter has about a 0.05micron pore size, and contains approximately 550 cm² surface area offilter media. An individual media tube typically has a diameter of about0.5 mm. In processing 130 ml of the regenerative cell composition,approximately 120 ml of additional saline may be added to thecomposition. The processing or filter time may be approximately 8minutes. The differential of the pressures on either side of the body ofthe hollow fiber tube (e.g., the pressure inside the lumen of the body,and outside the body) is considered the trans-membrane pressure. Thetrans-membrane pressure can range from about 1 mmHg to about 500 mmHgwith a preferred pressure being about 200 mmHg. The average nucleatedcell recovery and viability using hollow fiber filtration can beapproximately 80% of viable cells.

The amount of collagenase which is typically removed in such a systemequates to a three log reduction. For example if the initialconcentration of collagenase in the regenerative cell composition whichis transferred from the collection chamber to the processing chamber is0.078 U/ml the collagenase concentration of the final regenerative cellcomposition would be 0.00078 U/ml. The collagenase is removed in thehollow fiber filter, and the hollow fiber filter corresponds to thethird filter discussed above.

Processing chambers illustrating one or more cell filtration methodsdescribed above are shown in the Figures, particularly FIGS. 1-3. Withreference to FIGS. 1-3, between the processing chamber 30 and thefiltering chamber of the filter assembly 36, a pump may be provided,such as pump 34. In addition, vent and pressure sensors, such as vent32, and pressure sensor 39, may be provided in line with the processingchamber 30 and the filter assembly 36. Fittings for the output chamber50 may also be provided. These optional components (e.g., the pump 34,the vent 32, the pressure sensor 39, and the fittings for the outputchamber 50) may be provided between the processing chamber 30 and thefilter assembly 36 so that liquid contained in the processing chamber 30may flow to one or more of these optional components before flowingthrough the filter assembly 36. For example, liquid may flow through thepump 34 before it is passed to the filter assembly 36. Or, liquid maypass through the pressure sensor 39 before passing through the filterassembly to obtain a pre-filter liquid pressure in the system. Incertain situations, one or more of these components may also be providedas an element of the processing chamber 30, such as the vent 32 asillustrated in FIG. 6. In the illustrated embodiment, the pressuresensor 39 is in line to determine the pressure of the regenerative cellcomposition which is generated by the pump 34 as it enters the filteringchamber of the filter assembly 36. This construction can facilitatemonitoring of the trans-membrane pressure across the filter membrane.Additional saline or other buffer and washing solution can be added tothe regenerative cell composition to assist in the removal of unwantedproteins as the composition is being filtered through the filterassembly 36. This repeated washing can be performed multiple times toenhance the purity of the regenerative cells. In certain embodiments,the saline can be added at any step as deemed necessary to enhancefiltration.

In one specific embodiment, which is provided by way of example and notlimitation, the unwanted proteins and saline or other washing solutionis removed in the following manner. The composition with theregenerative cells, as well as collagen and connective tissue particlesor fragments, adipocytes, and collagenase, is cycled through a series offilters until a minimum volume is reached. The minimum volume is afunction of the total hold up volume of the system and somepredetermined constant. The hold up volume is the volume of liquid whichis contained in the tubing and conduits if all of the processingchambers are empty. In one embodiment, the minimum volume is 15 ml. Whenthe minimum volume is reached, a predetermined volume of washingsolution is introduced into the system to be mixed with the regenerativecell composition. This mixture of washing solution and the regenerativecell composition is then cycled through the filters until the minimumvolume is reached again. This cycle can be repeated multiple times toenhance the purity of the regenerative cells, or in other words, toincrease the ratio of regenerative cells in the composition to the othermaterials in the composition. See FIGS. 10 and 11.

After it has been determined that the regenerative cell composition hasbeen cleansed of unwanted proteins and concentrated sufficiently (inexemplary embodiments, minimum concentrations within a range of about1×10⁵ to about 1×10⁷ cells/ml can be used and, in a preferred embodimentthe minimum concentration can be about 1×10⁷ cells/ml), an outputchamber 50, such as an output bag, may be connected to an outlet port ofthe processing chamber 30 and/or the filter assembly 36, depending onthe specific embodiment. A vent, such as the vent 32, may then be openedto facilitate the output of the concentrated regenerative cells. In oneimplementation, this determination of when a minimum concentration hasbeen reached is made empirically after experiments have been run andprogrammed into the electronic controls of the device. The determinationcan be an input into the process of what is desired to yield, i.e., howmany stem/progenitor cells are desired, or range of cell concentration.Based on scientific data, a predefined amount of adipose tissue needs tobe obtained and placed into the system to achieve the desired output.With the vent 32 open, a pump, such as the pump 34, can function totransfer the concentrated regenerative cells into the output bag. In oneembodiment, the output bag 50 is similar to an empty blood bag which hasa tube with a fitting on one end. In a sterile fashion, the fitting onthe output bag may be attached to the outlet port, and the concentratedregenerative cells may be transferred to the output bag.

As illustrated in FIGS. 1-3, a vacuum pump 26 may be provided in thesystem 10 to change the pressure in the system, among other things. Forexample, the vacuum pump 26 may be coupled to the collection chamber 20via a conduit, such as conduit 12 b, to cause a decrease in pressurewithin the collection chamber 20. Vacuum pump 26 may also be coupled tothe processing chamber 30 by way of a conduit, such as conduit 12 g.Regarding the operation of vacuum pump 26 in connection with pump 34,two separate vacuum pumps or sources may be implemented, or a single onemay be implemented by using valves which direct the vacuum pull to thedifferent conduits that need it at specific points in the process. Inaddition, vacuum pump 26 may be coupled to the waste container 40 via aconduit, such as conduit 12 f.

With reference to FIGS. 10 and 11, the pressure generated by the vacuumpump 26 can be used to direct the flow of fluids, including theregenerative cells, through the conduits 12. This pressure can besupplied in multiple directions, for example, by automatically ormanually controlling the position of one or more valves 14 in the system10. The system 10 can be made to function properly with the use ofpositive pressure or through the use of negative pressure, orcombinations thereof. For instance, the regenerative cells can be pulledthrough the first and second filters described above into a soft sidedcontainer which is connected to the third filter. The soft-sidedcontainer can be in line (serial) connected ahead of the third filter.The final output chamber may be a soft sided container which is on theother side (e.g., the downstream side) of the third filter. In thisembodiment, pressure is used to move the regenerative cells from onesoft sided container to a second soft sided container through thefilter.

In another embodiment of the system 10, the filtration of the stem cellsand/or adipose derived progenitor cells may be accomplished using acombination of percolative filtration and sedimentation. For example,such a system uses saline that is passed through a tissue regenerativecell composition (e.g., the composition containing the stem cells and/oradipose derived progenitor cells) and then through a filter. Some of thevariables which are associated with percolative filtration of cells froma regenerative cell composition include, but are not limited to, poresize of the filter media, pore geometry or shape, surface area of thefilter, flow direction of the regenerative cell composition beingfiltered, flow rate of the infused saline, trans-membrane pressure,dilution of the cell population, cell size and viability.

In one embodiment of the system 10, the processing chamber 30 uses afilter assembly 36 which implements percolative filtration andsedimentation to separate and concentrate the regenerative cells. By wayof example, and not by way of limitation, the processing chamber 30 isdefined as a generally cylindrical body having a sidewall 30 a, a topsurface 30 b, and a bottom surface 30 c, as shown in FIG. 6. A sterilevent 32 is provided in the top surface 30 b.

In the embodiment of FIG. 6, the processing chamber 30 is illustrated asincluding a filter assembly 36, which includes two filters, such aslarge pore filter 36 a, and small pore filter 36 b. The pore sizes ofthe filters 36 a and 36 b typically are in a range between about 0.05microns and about 10 microns. The large pore filter 36 a may comprisepores with a diameter of about 5 μm, and the small pore filter 36 b maycomprise pores with a diameter of about 1-3 μm. In one embodiment, thefilters have a surface area of about 785 mm². Filters 36 a and 36 bdivide an interior of the processing chamber 30 to include a firstchamber 37 a, a second chamber 37 b, and a third chamber 37 c. As shownin FIG. 6, first chamber 37 a is located between second chamber 37 b andthird chamber 37 c. In addition, first chamber 37 a is shown as beingthe region of the processing chamber 30 having an inlet port 31 a and anoutlet port 31 b. The illustrated processing chamber 30 includes aplurality of ports providing communication paths from an exterior of theprocessing chamber 30 to the interior of the processing chamber 30, suchas ports 31 a, 31 b, and 31 c. The ports 31 a, 31 b, and 31 c, areillustrated as being disposed in the sidewall 30 a of a body of theprocessing chamber 30. However, the ports 31 a, 31 b, and 31 c could bepositioned in other regions, as well. Port 31 a is illustrated as asample inlet port, which is constructed to be coupled to a conduit sothat a composition containing regenerative cells can be passed into theinterior of the processing chamber 30. Port 31 b is illustrated as anoutlet port constructed to be coupled to a conduit so that the separatedand concentrated cells may be removed from the interior of theprocessing chamber 30. Port 31 c is illustrated as an inlet portconstructed to be coupled to a conduit for delivery of a fresh washingsolution, such as saline into the interior of the processing chamber 30.

In use, the regenerative cells may be introduced into the centralchamber 37 a via inlet port 31 a. Saline or other buffer is introducedinto the bottom chamber 37 b through inlet port 31 c. The saline may bedirected through the regenerative cell composition in chamber 37 a at arate of about 10 ml/min. The flow rate of the saline is such that itcounteracts the force of gravity. The flow of saline gives the cells inthe chamber the ability to separate based on the density of the cells.Typically, as the saline is forced up through the composition the largercells in the composition will settle to the bottom of the centralchamber 37 a, and the smaller cells and proteins will be carried awaythrough the second filter 36 b into the top chamber 37 c. This filteringis accomplished by adjusting the flow rate of the saline such that thelarger cells are rolled in place which allows the smaller particles tobe liberated and carried off with the saline. The sterile vent 32 isincluded in the chamber 30 to ensure that the correct pressure gradientis maintained in the three chambers within the processing unit. Theupper chamber 37 c can comprise an absorbent media 33. The purpose ofthe absorbent media is to trap the unwanted proteins in the solution toensure that they do not cross the filter media back into the processingsolution, if, for example, the saline flow rate decreases. An absorbentmedia can be a type of filter material that is absorbent, or attractsmaterials or components to be filtered out. An outflow port can be addedabove the top filter to help draw off the waste. Another embodiment ofthis may be to apply a gentle vacuum from the top to help pull offwaste. Absorbent media can be implemented when, as in the illustratedembodiment, the flow rates are relatively small. Excess saline andproteins are then carried away to a waste container.

When the larger cells, (e.g., the adipose derived stem cells and/orprogenitor cells) have been sufficiently separated from smaller cellsand proteins, the composition containing the separated cells may beconcentrated, as discussed herein. The composition may be furtherconcentrated after it has been removed from chamber 37 a through outletport 31 b, or while it is in the chamber 37 a. In one embodiment, theconcentration of cells in the composition is increased in the followingmanner. After the cells have been sufficiently separated the filters,such as filters 36 a and 36 b, may be moved towards each other. Thismovement has the effect of reducing the volume between the two filters(e.g., the volume of chamber 37 a). A vibrating member may also beprovided in connection with the processing chamber 30 to facilitateconcentrating of the cells in the composition. In one embodiment, thevibrating member may be coupled to the filter 36 b (e.g., the small porefilter). Vibrating can reduce an incidence of cells becoming trapped inthe filters. The reduction in volume of the composition allows theexcess saline to be removed as waste and the cells to be concentrated ina smaller volume.

In another embodiment, the concentration of the regenerative cells isaccomplished in the following manner. After the cells have beensufficiently separated, the regenerative cell composition can betransferred to another chamber (not shown) which uses gravity to filterout the excess saline. In a preferred embodiment, the sedimentation canoccur at the same time as the percolation. This sedimentation may beaccomplished by introducing the composition on top of a filter which hasa pore size ranging from about 10 kD to about 2 microns. In oneembodiment, a suitable filter has a pore size of about 1 micron. Theforce of gravity will allow the saline and smaller particles to bepassed through the filter while preventing the cells in the compositionto flow through the filter. After the desired concentration of cells hasbeen obtained, and after the filtered smaller particles have beenremoved from below the filter, the regenerative cell composition may beagitated to remove the cells from the filter and, subsequently, theconcentrated regenerative cells may be transferred to the output bag.The smaller particles can be drawn off as waste through an outlet.

In a particular embodiment, the regenerative cell composition from thecollection chamber 20 is transported to the processing chamber 30wherein the composition can be centrifuged to separate and concentrateregenerative cells. Centrifugation principles are well know in the artand will be not be repeated herein in the interest of brevity. Standard,art-recognized centrifugation devices, components and parameters areutilized herein. An exemplary processing chamber for use as part of acentrifuge device is shown in FIGS. 7 and 8. Typically, a centrifugedevice causes a centrifuge chamber (such as the one shown in FIG. 7) tospin around an axis to thereby increasing the force on the cells in thesolution to be greater than gravity. The denser or heavier materials inthe solution typically settle to one end of the centrifuge chamber,i.e., an output chamber 50 of FIG. 7, to form a regenerative cellpellet. The pellet may then be re-suspended to obtain a solution with adesired concentration of cells and/or a desired volume of cells andmedium. The processing chamber shown in FIG. 7 is constructed toseparate and concentrate cells using both centrifugal and gravitationalforces. Specifically, during centrifugation, centrifugal force directsthe denser components of the regenerative cell composition, e.g., theregenerative cells, towards the outermost ends of the centrifugechamber. As the centrifuge chamber slows down and eventually stops,gravitational force helps the regenerative cells to remain in theoutermost ends of the centrifuge chamber and form a cell pellet.Accordingly, the unwanted components of the regenerative cellcomposition, i.e., the waste, can be removed without disturbing the cellpellet.

In yet another embodiment of the invention, the processing chamber maybe comprised of a cell concentrator in the form of a spinning membranefilter. In a further embodiment of the centrifugation process,centrifugal elutriation may also be applied. In this embodiment, thecells may be separated based on the individual cell sedimentation ratesuch that the directional (e.g., outward) force applied bycentrifugation causes cells and solutes to sediment at different rates.In elutriation, the sedimentation rate of the target cell population isopposed by an opposite (e.g., inward) flow rate applied by pumpingsolution in the opposite direction to the centrifugal force. Thecounterflow is adjusted so that the cells and particles within thesolution are separated. Elutriation has been applied in many instancesof cell separation (Inoue, Carsten et al. 1981; Hayner, Braun et al.1984; Noga 1999) and the principles and practices used to optimize flowand centrifugal parameters can be applied herein in light of the presentdisclosure by one skilled in the art.

FIG. 9 illustrates principles associated with an elutriationimplementation in accordance with the present invention. The elutriationembodiment can be similar to a centrifugation implementation to theextent that a force is applied to the solution using a spinning rotor.Some of the variables which are associated with the presently embodiedelutriation separation include, but are not limited to, the size andshape of the spinning chamber, the diameter of the rotor, the speed ofthe rotor, the diameter of the counter flow tubing, the flow rate of thecounter flow, as well as the size and density of the particles and cellswhich are to be removed from solution. As in centrifugation, theregenerative cells can be separated based on individual cell densities.

In one embodiment the regenerative cell composition, e.g., the solutioncontaining the regenerative cells and the collagenase, is introducedinto a chamber of a spinning rotor, as shown in FIG. 9.1. After thesolution is added to the chamber additional saline is added to thechamber at a predetermined flow rate. The flow rate of the saline can bepredetermined as a function of the speed of the rotor, the celldiameter, and the chamber constant which has been establishedempirically. The flow rate will be controlled for example with a devicesimilar to an IV pump. A purpose of the additional saline is to providea condition inside the rotor chamber where the larger particles willmove to one side of the chamber and the smaller particles will move tothe other, as illustrated in FIG. 9.2. The flow is adjusted so that, inthis application, the smaller particles will exit the chamber and moveto a waste container, as shown in FIG. 9.3. This movement results in thesolution in the rotor chamber having a substantially homogenouspopulation of cells, such as stem cells. After it has been determinedthat the stem cells have been separated from the rest of the items inthe solution (with unwanted proteins and free lipids having been removedfrom the chamber), the counter flow is stopped. The cells inside thechamber will then form a concentrated pellet on the outside wall of thechamber. The counter flow is reversed and the cell pellet is transferredto the output bag.

As previously set forth herein, the processing chamber 30 or the outputchamber 50 may include one or more ports, e.g., ports 51 or 52. One ormore of these ports may be designed to transport the regenerative cellsobtained using any combination of methods described above, or a portionthereof, via conduits to other surgical devices, cell culturing devices,cell marinading devices, gene therapy devices or purification devices.These ports may also be designed to transport the regenerative cells viaconduits to additional chambers or containers within the system or aspart of another system for the same purposes described above. The portsand conduits may be also be used to add one or more additives, e.g.,growth factors, re-suspension fluids, cell culture reagents, cellexpansion reagents, cell preservation reagents or cell modificationreagents including agents that transfer genes to the cells. The portsand conduits may also be used to transport the regenerative cells toother targets such as implant materials (e.g., scaffolds or bonefragments) as well as other surgical implants and devices.

Further processing of the cells may also be initiated by reconfiguringthe interconnections of the disposable sets of the existing system,re-programming the processing device of the existing system, byproviding different or additional containers and/or chambers for theexisting system, by transporting the cells to a one or more additionalsystems or devices and/or any combinations thereof. For example, thesystem can be reconfigured by any of the means described above such thatthe regenerative cells obtained using the system may be subject to oneor more of the following: cell expansion (of one or more regenerativecell types) and cell maintenance (including cell sheet rinsing and mediachanging); sub-culturing; cell seeding; transient transfection(including seeding of transfected cells from bulk supply); harvesting(including enzymatic, non-enzymatic harvesting and harvesting bymechanical scraping); measuring cell viability; cell plating (e.g., onmicrotiter plates, including picking cells from individual wells forexpansion, expansion of cells into fresh wells); high throughputscreening; cell therapy applications; gene therapy applications; tissueengineering applications; therapeutic protein applications; viralvaccine applications; harvest of regenerative cells or supernatant forbanking or screening, measurement of cell growth, lysis, inoculation,infection or induction; generation of cells lines (including hybridomacells); culture of cells for permeability studies; cells for RNAi andviral resistance studies; cells for knock-out and transgenic animalstudies; affinity purification studies; structural biology applications;assay development and protein engineering applications.

For example, if expansion of a regenerative cell population is requiredfor a particular application, an approach using culture conditions topreferentially expand the population while other populations are eithermaintained (and thereby reduced by dilution with the growing selectedcells) or lost due to absence of required growth conditions could beused. Sekiya et al have described conditions which might be employed inthis regard for bone marrow-derived stem cells (Sekiya et al., 2002).This approach (with or without differential adherence to the tissueculture plastic) could be applied to a further embodiment of thisinvention. In this embodiment the final regenerative cell pellet isremoved from the output chamber and placed into a second systemproviding the cell culture component. This could be in the form of aconventional laboratory tissue culture incubator or a Bioreactor-styledevice such as that described by Tsao et al., U.S. Pat. No. 6,001,642,or by Armstrong et al., U.S. Pat. No. 6,238,908. In an alternativeembodiment, the cell expansion or cell culture component could be addedto the existing system, e.g., into the output chamber, allowing forshort-term adherence and/or cell culture of the adipose derived cellpopulations. This alternate embodiment would permit integration of thecell culture and/or cell expansion component to the system and removethe need for removing the cells from this system and placement withinanother.

During the processing, one or more additives may be added to or providedwith the various chambers or containers as needed to enhance theresults. These additives may also be provided as part of another systemassociated with the existing system or separate from the existingsystem. For example, in certain embodiments, the additives are added orprovided without the need for removing the regenerative cells from thesystem. In other embodiments, the additives are added or provided byconnecting a new container or chamber comprising the additives into anunused port of the system in a sterile manner. In yet other embodiments,the additives are added or provided in a second system or device that isnot connected to the system of the present invention. Some examples ofadditives include agents that optimize washing and disaggregation,additives that enhance the viability of the active cell populationduring processing, anti-microbial agents (e.g., antibiotics), additivesthat lyse adipocytes and/or red blood cells, or additives that enrichfor cell populations of interest (by differential adherence to solidphase moieties or to otherwise promote the substantial reduction orenrichment of cell populations) as described herein.

For example, to obtain a homogenous regenerative cell population, anysuitable method for separating and concentrating the particularregenerative cell type may be employed, such as the use of cell-specificantibodies that recognize and bind antigens present on, for example,stem cells or progenitor cells, e.g., endothelial precursor cells. Theseinclude both positive selection (selecting the target cells), negativeselection (selective removal of unwanted cells), or combinationsthereof. Intracellular markers such as enzymes may also be used inselection using molecules which fluoresce when acted upon by specificenzymes. In addition, a solid phase material with adhesive propertiesselected to allow for differential adherence and/or elution of aparticular population of regenerative cells within the final cell pelletcould be inserted into the output chamber of the system.

An alternate embodiment of this differential adherence approach wouldinclude use of antibodies and/or combinations of antibodies recognizingsurface molecules differentially expressed on target regenerative cellsand unwanted cells. Selection on the basis of expression of specificcell surface markers (or combinations thereof) is another commonlyapplied technique in which antibodies are attached (directly orindirectly) to a solid phase support structure (Geiselhart et al., 1996;Formanek et al., 1998; Graepler et al., 1998; Kobari et al., 2001; Mohret al., 2001).

In another embodiment the cell pellet could be re-suspended, layeredover (or under) a fluid material formed into a continuous ordiscontinuous density gradient and placed in a centrifuge for separationof cell populations on the basis of cell density. In a similarembodiment continuous flow approaches such as apheresis (Smith, 1997),and elutriation (with or without counter-current) (Lasch et al., 2000)(Ito and Shinomiya, 2001) may also be employed.

Other examples of additives may include additional biological orstructural components, such as cell differentiation factors, growthpromoters, immunosuppressive agents, medical devices, or anycombinations thereof, as discussed herein. For example, other cells,tissue, tissue fragments, growth factors such as VEGF and other knownangiogenic or arteriogenic growth factors, biologically active or inertcompounds, resorbable scaffolds, or other additives intended to enhancethe delivery, efficacy, tolerability, or function of the population ofregenerative cells may be added. The regenerative cell population mayalso be modified by insertion of DNA or by placement in a cell culturesystem (as described herein or known in the art) in such a way as tochange, enhance, or supplement the function of the regenerative cellsfor derivation of a structural or therapeutic purpose. For example, genetransfer techniques for stem cells are known by persons of ordinaryskill in the art, as disclosed in (Morizono et al., 2003; Mosca et al.,2000), and may include viral transfection techniques, and morespecifically, adeno-associated virus gene transfer techniques, asdisclosed in (Walther and Stein, 2000) and (Athanasopoulos et al.,2000). Non-viral based techniques may also be performed as disclosed in(Muramatsu et al., 1998). A gene encoding one or more cellulardifferentiating factors, e.g., a growth factor(s) or a cytokine(s),could also be added. Examples of various cell differentiation agents aredisclosed in (Gimble et al., 1995; Lennon et al., 1995; Majumdar et al.,1998; Caplan and Goldberg, 1999; Ohgushi and Caplan, 1999; Pittenger etal., 1999; Caplan and Bruder, 2001; Fukuda, 2001; Worster et al., 2001;Zuk et al., 2001). Genes encoding anti-apoptotic factors or agents couldalso be added. Addition of the gene (or combination of genes) could beby any technology known in the art including but not limited toadenoviral transduction, “gene guns,” liposome-mediated transduction,and retrovirus or lentivirus-mediated transduction, plasmid,adeno-associated virus. These regenerative cells could then be implantedalong with a carrier material bearing gene delivery vehicle capable ofreleasing and/or presenting genes to the cells over time such thattransduction can continue or be initiated in situ.

When the cells and/or tissue containing the cells are administered to apatient other than the patient from whom the cells and/or tissue wereobtained, one or more immunosuppressive agents may be administered tothe patient receiving the cells and/or tissue to reduce, and preferablyprevent, rejection of the transplant. As used herein, the term“immunosuppressive drug or agent” is intended to include pharmaceuticalagents which inhibit or interfere with normal immune function. Examplesof immunosuppressive agents suitable with the methods disclosed hereininclude agents that inhibit T-cell/B-cell costimulation pathways, suchas agents that interfere with the coupling of T-cells and B-cells viathe CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No.20020182211. A preferred immunosuppressive agent is cyclosporine A.Other examples include myophenylate mofetil, rapamicin, andanti-thymocyte globulin. In one embodiment, the immunosuppressive drugis administered with at least one other therapeutic agent. Theimmunosuppressive drug is administered in a formulation which iscompatible with the route of administration and is administered to asubject at a dosage sufficient to achieve the desired therapeuticeffect. In another embodiment, the immunosuppressive drug isadministered transiently for a sufficient time to induce tolerance tothe regenerative cells of the invention.

In these embodiments, the regenerative cells may be contacted, combined,mixed or added to the additives through any art recognized manner,including devices such as the agitation devices and associated methodsdescribed herein. For example, rocking, inversion, compression pulsed ormoving rollers may be used.

In another aspect, the cell population could be placed into therecipient and surrounded by a resorbable plastic sheath or othermaterials and related components such as those manufactured by MacroPoreBiosurgery, Inc. (see e.g., U.S. Pat. Nos. 6,269,716; 5,919,234;6,673,362; 6,635,064; 6,653,146; 6,391,059; 6,343,531; 6,280,473).

In all of the foregoing embodiments, at least a portion of the separatedand concentrated regenerative cells may be cryopreserved, as describedin U.S. patent application Ser. No. 10/242,094, entitled PRESERVATION OFNON EMBRYONIC CELLS FROM NON HEMATOPOIETIC TISSUES, filed Sep. 12, 2002,which claims the benefit of U.S. Provisional Patent Application60/322,070 filed Sep. 14, 2001, which is commonly assigned, and thecontents of which in their entireties are expressly incorporated hereinby reference.

At the end of processing, the regenerative cells may be manuallyretrieved from the output chamber. The cells may be loaded into adelivery device, such as a syringe, for placement into the recipient byeither, subcutaneous, intramuscular, or other technique allowingdelivery of the cells to the target site within the patient. In otherwords, cells may be placed into the patient by any means known topersons of ordinary skill in the art. Preferred embodiments includeplacement by needle or catheter, or by direct surgical implantation. Inother embodiments, the cells may be automatically transported to anoutput chamber which may be in the form of a container, syringe orcatheter etc., which may be used to place the cells in the patient. Thecontainer may also be used to store the cells for later use or forcryopreservation. All retrieval methods are performed in a sterilemanner. In the embodiment of surgical implantation, the cells could beapplied in association with additives such as a preformed matrix orscaffold as described herein.

In preferred embodiments of the invention (e.g., the embodiment shown inFIG. 4), the system is automated. In another embodiment, the system hasboth automated and manual components. The system may be comprised of oneor more disposable components connected to or mounted on a re-usablehardware component or module. The automated systems of the inventionprovide screen displays (see FIG. 16) that prompt proper operation ofthe system. The automated systems may also provide a screen thatprovides status of the procedure and/or the step by step instructions asto the proper setup of the disposable components of the system. Thescreen may also indicate problems or failures in the system if theyoccur and provide “troubleshooting” guidance if appropriate. In oneembodiment, the screen is a user interface screen that allows the userto input parameters into the system through, e.g., a touch screen.

The partial and fully automated systems may include a processing device(e.g., microprocessor or personal computer) and associated softwareprograms that provide the control logic for the system to operate and toautomate one or more steps of the process based on user input. Incertain embodiments, one or more aspects of the system may beuser-programmable via software residing in the processing device. Theprocessing device may have one or more pre-programmed software programsin Read Only Memory (ROM). For example, the processing device may havepre-programmed software tailored for processing blood, another programfor processing adipose tissue to obtain small volumes of regenerativecells and another program for processing adipose tissue to obtain largervolumes of regenerative cells. The processing device may also havepre-programmed software which provides the user with appropriateparameters to optimize the process based on the user's input of relevantinformation such as the amount of regenerative cells required, the typeof tissue being processed, the type of post-processing manipulationrequired, the type of therapeutic application, etc.

The software may also allow automation of steps such as controlling theingress and egress of fluids and tissues along particular tubing pathsby controlling pumps and valves of the system; controlling the propersequence and/or direction of activation; detecting blockages withpressure sensors; mixing mechanisms, measuring the amount of tissueand/or fluid to be moved along a particular pathway using volumetricmechanisms; maintaining temperatures of the various components usingheat control devices; and integrating the separation and concentrationprocess with timing and software mechanisms. The processing device canalso control centrifuge speeds based on the tissue type being processedand/or the cell population or sub-population being harvested, and thetypes of procedures to be performed (e.g., tissue enhancement usingadipose tissue augmented with regenerative cells, or processing of cellsfor bone repair applications using regenerative cell coated bonegrafts). The processing device may also include standard parallel orserial ports or other means of communicating with other computers ornetworks. Accordingly, the processing device can be a stand alone unitor be associated one or more additional devices for the furtherprocessing methods described herein.

The software may allow for automated collection of “run data” including,for example, the lot numbers of disposable components, temperature andvolume measurements, tissue volume and cell number parameters, dose ofenzyme applied, incubation time, operator identity, date and time,patient identity, etc. In a preferred embodiment of the device acharacter recognition system, such as a bar code reading system would beintegrated to permit data entry of these variables (for exampledisposable set lot number and expiration date, lot number and expirationdate of the Collagenase, patient/sample identifiers, etc.) into theprocessing device as part of documentation of processing. This wouldreduce the opportunity for data entry errors. Such a bar code readingsystem could be easily incorporated into the processing device using aUSB or other interface port and system known to the art. In this way thedevice would provide integrated control of the data entry anddocumentation of the process. A print-out report of these parameterswould be part of the user-defined parameters of a programmed operationof the system. Naturally this would require integration of a printercomponent (hardware and driver) or printer driver in software plus aninterface output connector for a printer (e.g., a USB port) in thehardware of the device.

In certain embodiments, the system is a fully automated system. Forexample, the user may initially select the amount of tissue to beprocessed, attach the system to the patient and the system mayautomatically aspirate the required tissue and separate and concentrateregenerative cells in an uninterrupted sequence without further userinput. The user may also input the amount of regenerative cells requiredand allow the system to aspirate the requisite amount of tissue andprocess the tissue. A fully automated system also includes a systemwhich is capable of being reconfigured based on a number of (e.g., twoor more) user input parameters, e.g., number of wash cycles, speed ofcentrifugation etc. The system can also be run in semi-automatic modeduring which the system goes through certain steps without userintervention but requires user intervention before certain processes canoccur. In other embodiments, the system is a single integrated systemthat displays instructions to guide the user to perform predeterminedoperations at predetermined times. For example, the processing devicemay prompt users through the steps necessary for proper insertion oftubing, chambers and other components of the system. Accordingly, theuser can ensure that the proper sequence of operations is beingperformed. Such a system can additionally require confirmation of eachoperational step by the user to prevent inadvertent activation ortermination of steps in the process. In a further embodiment, the systemmay initiate automated testing to confirm correct insertion of tubing,chambers, absence of blockages etc. In yet another embodiment, thesystem of the present invention is capable of being programmed toperform multiple separation and concentration processes throughautomated control of tissue flow through the system. This feature may beimportant, for example, during surgery on a patient where tissue thatwould otherwise be lost is collected into the system, and regenerativecells from the tissue are separated and concentrated and returned to thepatient.

As set forth above, components of the system may be disposable (referredto herein as “disposable set(s)”), such that portions of the system canbe disposed of after a single use. This implementation can help ensurethat any surface which comes in contact with the patient's tissue willbe disposed of properly after being used. An exemplary disposable set isillustrated in FIG. 13. In a preferred embodiment, the disposablecomponents of the system are pre-sterilized and packaged so as to beusable “off the shelf” that are easy to use and easy to load and thateliminate the need for many tubing connections and complex routing oftubing connections. Such disposable components are relativelyinexpensive to manufacture, and therefore, do not create a substantialexpense due to their disposal. In one embodiment, the disposable system(referred to interchangeably herein as “disposable set(s)”) comprises,consists essentially of, or consists of, the collection chamber 20, theprocessing chamber 30, the waste chamber 40, the output chamber 50, thefilter assemblies 36, the sample bag 60 and the associated conduits 12or tubing. In preferred embodiments of the disposable sets of thesystem, the collection chamber 20 and the processing chamber 30 areconnected by way of conduits 12 that are housed in a rigid frame. Therotating seal network (FIGS. 7 & 8) of a processing chamber 30 may alsobe housed in the same rigid frame. In another preferred embodiment, thevarious chambers and containers of the disposable set are comprised ofthe necessary interfaces that are capable of communicating with theprocessing device of the system such that the pumps, valves, sensors andother devices that automate the system are appropriately activated orde-activated as needed without user intervention. The interfaces alsoreduce the time and expertise required to set up the system and alsoreduce errors by indicating how to properly set up the system andalerting the user in the event of an erroneous setup.

Most of the disposable sets of the invention will have many commonelements. However, the ordinarily skilled artisan will recognize thatdifferent applications of the system may require additional componentswhich may be part of the disposable sets. Accordingly, the disposablesets may further comprise one or more needles or syringes suitable forobtaining adipose or other tissue from the patient and returningregenerative cells to the patient. The type number and variety of theneedles and syringes included will depend on the type and amount oftissue being processed. The disposable sets may further comprise one ormore rigid or flexible containers to hold washing fluids and otherprocessing reagents used in the system. For example, the disposable setsmay comprise containers to hold saline, enzymes and any other treatmentor replacement fluids required. for the procedure. In addition, suitablewashing solutions, re-suspension fluids, additives, agents or transplantmaterials may be provided with the disposable sets for use inconjunction with the systems and methods of the invention.

Any combination of system components, equipment or supplies describedherein or otherwise required to practice the invention may be providedin the form of a kit. For example, a kit of the invention may include,e.g., the optimal length and gage needle for the syringe basedliposuction and sterile syringes which contain the preferred filtermedia which allows for the processing of small volumes of tissue. Otherexemplary equipment and supplies which may be used with the inventionand may also be included with the kits of the invention are listed inTables II and III.

Table II below identifies examples of supplies that can be used in toobtain adipose derived regenerative cell in accordance with the systemsand methods of the present invention:

TABLE II Description Vendor Quantity Note 10 ml syringe Becton-Dickinsonas req'd Optional, used for liposuction 14 GA blunt tip needle as req'dOptional, used for liposuction Single Blood Pack Baxter Fenwal 1 Maincell processing bag; bag has (600 ml) spike adaptor on line and two freespike ports Transfer pack with coupler Baxter Fenwal 1 Quad bag set (150ml) Transfer pack with coupler Baxter Fenwal 1 Waste bag (1 L) SampleSite Coupler Baxter Fenwal 2 0.9% saline (for injection) Baxter Fenwal 114 GA sharp needle Monoject as req'd For adding liposuction tissue tobag 20 GA sharp needle Monoject 3 For adding collagenase and removingPLA cells 0.2 μm Sterflip filter Millipore 1 For filtering collagenaseTeruflex Aluminum sealing Terumo 4 ME*ACS121 for temporary tube clipssealing Povidone Iodine prep pad Triadine as req'd 10-3201 Liberase H1Collagenase Roche See Procedure Note 1 TSCD wafers Terumo 2 ISC*W017 foruse with TSCD

Table III, below, identifies equipment that may be used with the systemsand methods disclosed herein.

TABLE III Description Vendor Quantity Note Sorvall Legend T Easy SetFisher Scientific 1 75-004-367 Centrifuge Rotor Kendro/Sorvall 1 TTH-750rotor Rotor buckets Kendro/Sorvall 4 75006441 round buckets Adapter for150 ml bags Kendro/Sorvall 4 00511 Plasma Expressor Baxter Fenwall 14R4414 Tube Sealer Sebra 1 Model 1060 TSCD Sterile Tubing Terumo 13ME*SC201AD Welder Labline Thermal Rocker LabLine 1 4637 ‘Disposable’plastic Davron 3 hemostat-style clamp Balance Bags Sets 2 Water-filledbags used to balance centrifuge Biohazard Sharps Chamber 1 BiohazardWaste Chamber 1

The re-usable component of the system comprises, consists essentiallyof, or consists of the agitation mechanism for the collection chamber,the pump, and assorted sensors which activate valves and pump controls,the centrifuge motor, the rotating frame of the centrifuge motor, theuser interface screen and USB ports, an interlocking or docking deviceor configuration to connect the disposable set such that the disposableset is securely attached to and interface with the re-usable hardwarecomponent and other associated devices. An exemplary re-usable componentis illustrated in FIG. 14. In preferred embodiments, the re-usablecomponent includes a means for separating and concentrating theregenerative cells from the regenerative cell composition, e.g., arotating centrifuge. In this embodiment, the re-usable component isdesigned connect to and interface with a portion of the processingchamber (comprising a centrifuge chamber) of the disposable set as shownin FIG. 15A. It is understood that the means for separating andconcentrating regenerative cells in the re-usable component is notlimited to a rotating centrifuge but may also include any otherconfiguration described herein, including a spinning membrane filter.The re-usable component may also house the processing device describedherein which contains pre-programmed software for carrying out severaldifferent tissue processing procedures and selectively activating thevarious pumps and valves of the system accordingly. The processor mayalso include data storage capability for storing donor/patientinformation, processing or collection information and other data forlater downloading or compilation. The re-usable component may be usedwith a variety of disposable sets. The disposable set is connected tothe re-usable component through, e.g., an interlocking device orconfiguration to connect the disposable set such that the disposable setis securely attached to and interfaces with the re-usable hardwarecomponent in a manner that the processing device present on there-usable component can control, i.e., send and receive signals to andfrom the various components of the disposable set as well as variouscomponents of the re-usable component and other associated devices andsystems.

In one embodiment, a disposable set for use in the system is comprisedof a collection chamber 20 which can accommodate about 800 mL of tissue;a processing chamber 30 which can process the regenerative cellcomposition generated by about 800 mL of tissue washed and digested inthe collection chamber 20; an output chamber 50 which can accommodate atleast 0.5 mL of regenerative cells; and a waster container 40 which canaccommodate about 10 L of waste. In this embodiment, the hardware deviceis no larger than 24″L×18″W×36″H. Alternative dimensions of the variouscomponents of the disposable sets as well as the hardware device may beconstructed as needed and are intended to be encompassed by the presentinvention without limitation.

The disposable components of the system are easy to place on the device.An illustration of a disposable set utilized assembled together with acorresponding re-usable component is illustrated in FIG. 15A. The systemis preferably designed such that it can detect an improperly loadeddisposable component. For example, the components of each disposable setmay have color-guided marks to properly align and insert the tubing,chambers etc. into appropriate places in the system. In additionalembodiments, the system disclosed herein is a portable unit. Forexample, the portable unit may be able to be moved from one locationwhere adipose tissue harvesting has occurred, to another location foradipose tissue harvesting. In certain implementations, the portable unitis suitable for harvesting and processing of adipose tissue by apatient's bedside. Thus, a portable unit may be part of a system whichcan be moved from patient to patient. Accordingly, the portable unit maybe on wheels which lock in place and, thus, can be easily placed andused in a convenient location in a stable and secure position throughoutthe procedure. In other embodiments, the portable unit is designed forset-up and operation on a flat surface such as a table top. The portableunit may also be enclosed in a housing unit. The portable unit mayfurther be comprised of hangers, hooks, labels, scales and other devicesto assist in the procedure. All of the herein described re-usablecomponents of the system such as the centrifuge, processing device,display screen may be mounted on the portable unit of the system.

Alternate manual embodiments for obtaining regenerative cells are alsowithin the scope of this invention. For example, in one embodiment,adipose tissue may be processed using any combination of the componentsof the system, equipment and/or supplies described herein.

A manual embodiment of the system of the invention may be practiced inaccordance with the following steps and information, which are providedby way of example and not by way of limitation. First, adipose tissue iscollected from a patient. A tissue retrieval line, or sampling sitecoupler, is opened and a spike is inserted into a side port of the 600ml blood bag. Approximately 10 ml of adipose tissue is collected in a 10ml syringe through the blunt cannula. The blunt cannula is replaced witha relatively sharp needle (14G). The sampling site is wiped with aniodine wipe. The adipose tissue is injected into the 600 ml bag throughthe sampling site. The syringe and needle are then discarded in a sharpschamber. These steps are repeated to place sufficient tissue into thebag. Sufficient tissue is determined on a case-by case basis based onthe clinical specifics of the patient and application.

Second, the aspirated adipose tissue is washed. A pre-warmed (37° C.)saline bag is hooked above the work surface. A blue hempostat clamp isplaced on the tubing between the 600 ml bag and the spike. The clamp isclosed to seal the tubing. The spike on the 600 ml bag is used to enterthe saline bag (in this setting use the needle on the 600 ml bag toenter the saline bag through the rubber septum, wipe the septum withiodine prior to insertion of needle). The blue clamp is released andapproximately 150 ml of saline is allowed to enter the 600 ml bag. Theblue clamp is closed when the desired volume of saline has entered the600 ml bag. The 600 ml bag is inverted 10-15 times over approximately 15seconds. A second blue clamp is applied to the tubing leading from the 3L waste bag to the spike. The spike on the 3 L bag is used to enter the600 ml bag. The 600 ml bag is hung inverted over the work surface, andis allowed to sit for approximately 1 minute. The blue clamp leading tothe 3 L bag is released. Waste fluid is allowed to flow into the 3 Lbag. The blue clamp is applied to stop the flow before tissue enters thetubing. The 600 ml bag is lowered to the work surface. These steps arerepeated two more times. If the saline waste still appears noticeablyred, a third additional cycle is indicated. A heat sealer is used toseal the tubing between the 3 L waste bag and the 600 ml bag. The sealis made at approximately the half way point on the tubing. The 3 L wastebag is removed and discarded. The 600 ml bag is returned to the worksurface.

Third, the washed adipose tissue is digested. The blue clamp on thetubing between the saline and the 600 ml bag is released to allowapproximately 150 ml of saline to enter the 600 ml bag. The samplingsite on the 600 ml bag is wiped with an iodine wipe. Collagenase isinjected through the sampling site to the 600 ml bag. The collagenase isprepared by thawing one collagenase vial in a 37° C. water bath orequivalent, other than microwaving. A 1 ml syringe with a 22G needle isinserted into the vial. The collagenase is withdrawn into the needle.The needle is removed and replaced with a 0.2 μm filter and second 22Gneedle. The collagenase is then expelled from the syringe through the0.2 μm filter and needle. Digestion of the adipose tissue is performedat a final collagenase concentration of 0.1-0.2 Wunsch units/ml. Theheating pad is placed on the rocker. During this time, the saline bag,while still attached, is set to the side of the rocker. Care is taken toensure that the tubing leading to the saline bag is positioned in such away that it does not get caught on the rocker when in motion. Theheating pad controller is set to 37° C. The 600 ml bag is placed on therocker. The rocker is set to maximum. The bag is observed to ensure thatit is stable, and is allowed to rock for approximately 1 hour (55.+−.10mins).

Fourth, the regenerative cell composition is retrieved. The bag isremoved from the rocker. A blue clamp is applied to the closed tubingformerly leading to the waste bag. The sterile connecting device is usedto attach the quad bag set (pre-prepared according to the followinginstructions) to the tubing that was formerly attached to the waste bag.The quad pack can be seen as two linked quad packs. Identify the tubingsplitting it into two packs, fold the tubing back on itself, and slip ametal loop over the folded tubing (over both pieces of tubing). Slidethe loop down approx 0.5 inch. The crimp formed at the bend acts to sealthe tubing. Use a hemostat to partially crimp the loop closed. The loopis not crimped too tightly because the loop will need to be removedduring processing. The 600 ml bag is hung inverted over the work surfaceand is allowed to sit for approximately 3 minutes. The blue clamp ontubing leading to the quad set is released to drain the cell fraction(the layer under the yellow/orange fat layer) into the quad set. Care istaken to prevent the fat layer to enter the tubing. During this process,the tubing can be crimped manually to slow the flow as the fat layergets close to the tubing. The tubing leading to the quad bag set is thenclosed with a blue clamp, the 600 ml bag is returned to the worksurface, and the saline bag is hung. The blue clamp on the tubingbetween the saline and the 600 ml bag is released to allow approximately150 ml of saline to enter the 600 ml bag. The 600 ml bag is invertedapproximately 10-15 times over approximately 15 seconds. The 600 ml bagis then hung inverted over the work surface and is allowed to sit forabout 3-5 minutes. The blue clamp on tubing leading to the quad set isreleased, and the cell fraction (the layer under the yellow/orange fatlayer) is drained into the quad set. Care is taken to prevent the fatlayer from entering the tubing. For example, the flow can be slowed asthe fat layer gets close to the tubing by crimping the tubing manually.The tubing leading to the quad bag set is closed with a blue clamp. Thetubing leading from the quad set to the 600 ml bag is then heat sealed.The 600 ml bag is then removed and discarded.

Fifth, the regenerative cell composition is washed. A metal clip isplaced on the tubing between the two full bags to seal the tubing. Thequad set is placed on a balance. Water is added to a second “dummy” quadset to balance the quad set. The quad set and balanced set are placed onopposite buckets of the centrifuge. For the hollow filter, the cells arewashed and placed in the bag, and tubing is sealed between the bag andthe hollow fiber filter assembly described above. Using a peristalticpump, the fluid is run through the filter assembly and the cellconcentrate is collected in a bag on the downstream end. Care is takento make sure the quad set bags are not compressed and are upright. Thecentrifuge is operated at 400×g for 10 minutes. The quad set is removedfrom the centrifuge and placed in the plasma expressor. Care is taken toplace the bags in the expressor in such a way that the hard tubing atthe top of the bag is just at the top of the backplate. If the bag istoo high, too much saline will be retained, if it is too low the tubingwill interfere with the front plate's ability to close and again toomuch saline will be retained. A blue clamp is applied to each of thelines leading from the full quad set to the empty one. The metal loopsand blue clamps are removed to allow supernatant to flow into the emptyquad set. As much saline as possible is expressed off, but care is takennot to dislodge the cell pellet. The tubing running into each of thebags containing supernatant is heat sealed. The waste bags containingthe supernatant are removed. Blue clamps are applied to the tubingleading to each of the quad set bags containing cells. The bags aretaken out of the plasma expressor. A sterile connecting device is usedto connect the tubing leading to the quad pack to the saline bag. Theblue clamp leading to one of the quad set bags is removed to allowapproximately 150 ml saline to flow into the bag, and then the clamp isreapplied to stop the flow of saline. The full quad set bag is theninverted approximately 10-15 times for approximately 15 seconds. Theblue clamp leading to the empty quad set bag is then removed and all ofthe contents of full bag are drained into the empty bag. The metal loopclamp is reapplied to seal the tubing between two quad set bags. Thetubing is then heat sealed and the saline bag is removed. The full quadset bag is then inverted approximately 10-15 times over approximately 15seconds. Another dummy quad set is placed on a balance and isre-balanced to the cell quad set. The quad set bags (one full, oneempty) are then placed into the centrifuge so that the quad set bags arenot compressed and are upright.

The centrifuge is operated at about 400×g for 10 minutes. The quad setis then removed from the centrifuge and is placed carefully in theplasma expressor in such a way that the hard tubing at the top of thebag is just at the top of the backplate. If the bag is too high too muchsaline will be retained, if it is too low the tubing will interfere withthe front plate's ability to close and again too much saline will beretained. The metal loop is removed to express all the supernatant fromthe full bag into the empty bag taking care not to dislodge theregenerative cell pellet. The tubing between the bags is sealed, and thefull (waste) bag is removed and discarded. A new sampling site coupleris then inserted into the remaining bag. The cells of the cell pelletare then resuspended in the residual saline (if any) to obtain aconcentration of regenerative cells. The resuspension can be performedby gentle manipulation of the bag (e.g., squeezing and rubbing).

A particular example of the system embodying the present invention isshown in FIG. 4. FIG. 4 illustrates an automated system and method forseparating and concentrating regenerative cells from tissue, e.g.,adipose tissue, suitable for re-infusion within a patient. In certainembodiments of the system shown in FIG. 4, the system further includesan automated step for aspirating a given amount of tissue from thepatient. The system shown in FIG. 4 is comprised of the disposable setshown in FIG. 13 which is connected to the re-usable component of thesystem shown in FIG. 14 to arrive at an automated embodiment of thesystem shown in FIG. 15A. The disposable set is connected to there-usable component through, e.g., an interlocking or docking device orconfiguration, which connects the disposable set to the re-usablecomponent such that the disposable set is securely attached to andassociated with the re-usable hardware component in a manner that theprocessing device present on the re-usable component can control andinterface with, i.e., send and receive signals to and from the variouscomponents of the disposable set as well as various components of there-usable component and other associated devices and systems.

The user may connect the disposable set to the re-usable component,input certain parameters using the user interface, e.g., the volume oftissue being collected, attach the system to the patient, and the systemautomatically performs all of the steps shown in FIG. 4 in anuninterrupted sequence using pre-programmed and/or user inputparameters. One such sequence is illustrated in FIG. 15B. Alternatively,the tissue may be manually aspirated from the patient by the user andtransported to system for processing, i.e., separation and concentrationof regenerative cells.

Specifically, as shown in FIG. 4, tissue, e.g., adipose tissue, may bewithdrawn from the patient using conduit 12 and introduced intocollection chamber 20. A detailed illustration of the collection chamberof FIG. 4 is shown in FIG. 5. As illustrated in FIG. 5, the collectionchamber 20 may be comprised of a vacuum line 11 which facilitates tissueremoval using a standard cannula. The user may enter the estimatedvolume of tissue directed to the collection chamber 20 at this point.The tissue is introduced into the collection chamber 20 through an inletport 21 which is part of a closed fluid pathway that allows the tissue,saline and other agents to be added to the tissue in an aseptic manner.An optical sensor of the system, e.g., sensor 29, can detect when theuser input volume of tissue is present in the collection chamber 20. Incertain embodiments, if less tissue is present in the collection chamberthan the user input, the user will have the option to begin processingthe volume of tissue which is present in the collection chamber 20. Incertain embodiments, a portion of the tissue removed from the patientmay be directed to the sample chamber 60 through the use of a pump,e.g., a peristaltic pump, via a conduit, which may be activated via userinput utilizing the user interface.

A sensor 29 can signal the processing device present in the re-usablecomponent to activate the steps needed to wash and disaggregate thetissue. For example, the processing device may introduce a pre-setvolume of washing agent based on the volume of tissue collected usingautomated valves and pumps. This cycle may be repeated in the collectionchamber until the optical sensor determines that the effluent liquid issufficiently clear and devoid of unwanted material. For example, anoptical sensor 29 along the conduit leading out of the collectionchamber 12 b or 12 d can detect that the unwanted materials have beenremoved and can signal the processing device to close the requiredvalves and initiate the next step.

Next, the processing device may introduce a pre-programmed amount ofdisaggregation agent based on the volume of tissue collected. Theprocessing device may also activate agitation of the tissue in thecollection chamber for a preset period of time based on the initialvolume of tissue collected or based on user input. In the embodimentshown in FIG. 4, once the disaggregation agent, e.g., collagenase, isadded to the collection chamber 20 through the collagenase source 24,the motor in the collection chamber 20 is activated via the processingdevice. The motor activates the rotatable shaft 25 which is comprised ofa magnetic stirrer and a paddle-like device wherein one or more paddles25 a are rigidly attached to the filter cage 27 of a filter prefixed tothe collection chamber 28. The paddles agitate the in the presence ofthe disaggregation agent such that the regenerative cells separate fromthe tissue.

The solution in the collection chamber 20 is allowed to settle for apreset period of time. The buoyant portion of the solution is allowed torise to the top of the solution. Once the preset period of time elapses,the necessary valves and pumps are activated by the processing device toremove the non-buoyant portion to the waste chamber 40. The transferinto the waste chamber 40 continues until a sensor 29 along the conduitleading out of the collection chamber 12 b or 12 d can detect that thebuoyant fraction of the solution is about to be transferred to the wastechamber 30. For example, a sensor 29 along the conduit leading out ofthe collection chamber 12 b or 12 d can detect that the unwantedmaterials have been removed and can signal the processing device toclose the required valves.

At this time the non-buoyant fraction of the solution, i.e., theregenerative cell composition, is moved to the processing chamber 30.This is accomplished through the use of the necessary valves andperistaltic pumps. In certain embodiments, before transfer of theregenerative cell composition to the processing chamber 30, anadditional volume of saline may be added to the buoyant fraction ofsolution remaining in the collection chamber 20. Another wash cycle maybe repeated. After this cycle, the solution is allowed to settle and thenon-buoyant fraction (which contains the regenerative cells) istransported to the processing chamber 30 and the buoyant fraction isdrained to the waste chamber 40. The additional wash cycle is used tooptimize transfer of all the separated regenerative cells to theprocessing chamber 30.

Once the regenerative cell composition is transported to the processingchamber 30 by way of conduits 12, the composition may be subject to oneor more additional washing steps prior to the start of the concentrationphase. This ensures removal of waste and residual contaminants from thecollection chamber 20. Similarly, subsequent to the concentration step,the regenerative cell composition may be subjected to one or moreadditional washing steps to remove residual contaminants. The unwantedmaterials may be removed from the processing chamber 30 to the wastechamber 40 in the same manner, i.e., control of valves and pumps viasignals from the processing device, as described above.

The various embodiments of the processing chamber 30 shown in FIG. 4 aredescribed in detail below. The processing chamber 30 shown in FIG. 4 isin the form of a centrifuge chamber. A detailed illustration of theprocessing chamber of FIG. 4 is shown in FIGS. 7 and 8. Such aprocessing chamber 30 is generally comprised of a rotating seal network30.1 comprising an outer housing 30.2, one or more seals 30.3, one ormore bearings 30.4 and an attachment point 30.6 for connecting theprocessing chamber to the centrifuge device present in the re-usablecomponent of the system; one or more fluid paths 30.5 in the form ofconduits extending out from the rotating seal and ending in a centrifugechamber on each end which is in the form of an output chamber 50 housedin a frame 53 wherein the frame is comprised of one or more ports 52 andone or more handles to manually re-position the output chamber 50.

The rotating seal network 30.1 is included to ensure that the fluidpathways of the processing chamber can be maintained in a sterilecondition. In addition, the fluid pathways of the processing chamber canbe accessed in a sterile manner (e.g., to add agents or washingsolution) at any time, even while the centrifuge chamber of theprocessing chamber is spinning.

The rotating seal network 30.1 shown in FIGS. 7 and 8 includes arotating shaft comprised of two or more bearings 30.4, three or more lipseals 30.3, and an outer housing 30.2. In this embodiment, the bearings30.4 further comprise an outer and inner shaft (not shown) referred toherein as races. These races may be separated by precision groundspheres. The races and spheres comprising the bearings are preferablyfabricated with material suitable for contact with bodily fluid, or arecoated with material suitable for contact with bodily fluid. In apreferred embodiment, the races and spheres are fabricated using, forexample, silicone nitride or zirconia. Furthermore, in this embodiment,the three lip seals are comprised of a circular “U” shaped channel (notshown) as well as a circular spring (not shown). The circular “U” shapedchannel is preferably fabricated using flexible material such that aleakage proof junction with the rotating shaft of the rotating sealnetwork 30.1 is formed. Additionally, the lip seals are preferablyoriented in a manner such that pressure from the regenerative cellcomposition flowing through the processing chamber causes the sealassembly to tighten its junction with the rotating shaft by way ofincreased tension. The seals may be secured in position by way of one ormore circular clips (not shown) which are capable of expanding and/orcollapsing as needed in order to engage a groove in the outer housing30.2 of the rotating seal network 30.1. The heat generated by or nearthe rotating seal network 30.1 must be controlled to prevent lysis ofthe cells in the solution which is being moved through the passage. Thismay be accomplished by, for example, selecting a hard material forconstructing the rotating shaft, polishing the area of the rotatingshaft which comes in contact with the seals and minimizing contactbetween the rotating shaft and the seal.

In another embodiment the rotating seal network 30.1 is comprised of asingle rubber seal 30.3 and an air gasket (not shown). This seal andgasket provide a tortuous path for any biologic matter which couldcompromise the sterility of the system. In another embodiment therotating seal network 30.1 is comprised of multiple spring loaded seals30.3 which isolate the individual fluid paths. The seals 30.3 arefabricated of a material which can be sterilized as well as seal therotating shaft without lubricant. In another embodiment the rotatingseal network 30.1 is compromised of a pair of ceramic disks (not shown)which create the different fluid paths and can withstand the rotation ofthe system and not cause cell lysis. In another embodiment the fluidpathway is flexible and is allowed to wind and unwind with respect tothe processing chamber. This is accomplished by having the flexiblefluid pathway rotate one revolution for every two revolutions of theprocessing chamber 30. This eliminates the need for a rotating sealaltogether.

The regenerative cell composition is pumped from the collection chamber20 along a fluid path through the axis of rotation of the rotating sealnetwork 30.1 and then divides into a minimum of two fluid pathways 30.5each of which radiate outward from the central axis of the processingchamber 30 and terminate near the outer ends of the processing chamber30, i.e., within the centrifuge chambers which house the output chambers50 (FIGS. 7 and 8). Accordingly, in a preferred embodiment, theprocessing chamber 30 is comprised of two or more output chambers 50 asshown in FIGS. 7 and 8. These output chambers 50 are positioned suchthat they are in one orientation during processing 30.7 and anotherorientation for retrieval of concentrated regenerative cells 30.8. Forexample, the output changes are tilted in one angle during processingand another angle for cell retrieval. The cell retrieval angle is morevertical than the processing angle. The two positions of the outputchamber 50 may be manually manipulated through a handle 53 whichprotrudes out of the processing chamber 30. The regenerative cells canbe manually retrieved from the output chambers 50 when they are in theretrieval orientation 30.8 using a syringe. In another embodiment, fluidpath 30.5 is constructed such that it splits outside the processingchamber and then connects to the outer ends of the processing chamber30, i.e., within the centrifuge chambers which house the output chambers50 (not shown). In this embodiment, large volumes of regenerative cellcomposition and/or additives, solutions etc. may be transported to thecentrifuge chamber and/or the output chambers directly.

With reference to FIGS. 4 and 7-9, between the collection chamber 20 andthe processing chamber 30, a pump 34 and one or more valves 14 may beprovided. In a preferred embodiment, the valves 14 are electromechanicalvalves. In addition, sensors, such as pressure sensor 29, may beprovided in line with the processing chamber 30 and the collectionchamber 20. The valves, pumps and sensors act in concert with theprocessing device present on the re-usable component (FIG. 14) toautomate the concentration steps of the system.

The sensors detect the presence of the regenerative cell composition inthe centrifuge chambers and activate the centrifuge device throughcommunication with the processing device of the system. The regenerativecell composition is then subjected to a pre-programmed load for apre-programmed time based on the amount of tissue originally collectedand/or user input. In certain embodiments, this step may be repeatedeither automatically or through user input. For example, the compositionis subjected to a load of approximately 400 times the force of gravityfor a period of approximately 5 minutes. The output chamber 50 isconstructed such that the outer extremes of the chamber form a smallreservoir for the dense particles and cells. The output chamber 50retains the dense particles in what is termed a ‘cell pellet’, whileallowing the lighter supernatant to be removed through a fluid path,e.g., a fluid path which is along the axis of rotation of the rotatingseal network 30.1 and travels from the low point in the center of theprocessing chamber 30 through the rotating seal network 30.1 to thewaste container 40. The valves 14 and pumps 34 signal the processingdevice to activate steps to remove the supernatant to the wastecontainer 40 without disturbing the cell pellet present in the outputchamber 50.

The cell pellet that is obtained using the system shown in FIG. 4comprises the concentrated regenerative cells of the invention. In someembodiments, after the supernatant is removed and directed to the wastechamber 40, a fluid path 30.5 may be used to re-suspend the cell pelletthat is formed after centrifugation with additional solutions and/orother additives. Re-suspension of the cell pellet in this manner allowsfor further washing of the regenerative cells to remove unwantedproteins and chemical compounds as well as increasing the flow of oxygento the cells. The resulting suspension may be subjected to another loadof approximately 400 times the force of gravity for another period ofapproximately 5 minutes. After a second cell pellet is formed, and theresulting supernatant is removed to the waste chamber 40, a final washin the manner described above may be performed with saline or some otherappropriate buffer solution. This repeated washing can be performedmultiple times to enhance the purity of the regenerative cell solution.In certain embodiments, the saline can be added at any step as deemednecessary to enhance processing. The concentrations of regenerativecells obtained using the system shown in FIG. 4 may vary depending onamount of tissue collected, patient age, patient profile etc. Exemplaryyields are provided in Table I.

The final pellet present in the output chamber 50 may then be retrievedin an aseptic manner using an appropriate syringe after the outputchamber 50 is positioned in the orientation appropriate for cellremoval. In other embodiments, the final pellet may be automaticallymoved to a container in the in the output chamber 50 which may beremoved and stored or used as needed. This container may be in anyappropriate form or size. For example, the container may be a syringe.In certain embodiments, the output container 50 itself may be heatsealed (either automatically or manually) and isolated from the othercomponents of the processing chamber for subsequent retrieval and use ofthe regenerative cells in therapeutic applications as described hereinincluding re-infusion into the patient. The cells may also be subject tofurther processing as described herein either prior to retrieval fromthe output chamber or after transfer to a second system or device. There-usable component shown in FIG. 14 is constructed such that it can beconnected to one or more additional systems or devices for furtherprocessing as needed.

As described herein, the regenerative cells obtained using the systemsand methods of the present invention can be used for wound healing basedon their properties as described in the Examples. For example, theregenerative cells have the ability to express angiogenic growth factorsand cytokines, secrete wound healing related cytokines, secrete collagenin vitro, and promote wound healing in vivo. Accordingly, in one aspectof the present invention, regenerative cells are extracted from adonor's adipose tissue using the systems and methods of the presentinvention and are used to heal a wound(s) through one or more of themechanisms demonstrated herein. In a preferred embodiment the cells areextracted from the adipose tissue of the person into whom they are to beimplanted, thereby reducing potential complications associated withantigenic and/or immunogenic responses to the transplant. Patients aretypically evaluated to assess a wound healing disorder by, e.g.,physical examination and the patient's history.

In one embodiment, the harvesting procedure is performed prior to thepatient receiving any products designed to reduce blood clotting.However, in certain embodiments, the patient may have received aspirinprior to the harvesting procedure. In addition, one preferred methodincludes collection of adipose tissue prior to any attempted procedure.However, as understood by persons skilled in the art, the timing ofcollection is expected to vary and will depend on several factorsincluding, among other things, patient stability, patient coagulationprofile, provider availability, and quality care standards. Ultimately,the timing of collection will be determined by the practitionerresponsible for administering care to the affected patient.

The volume of adipose tissue collected from the patient can vary fromabout 0 cc to about 2000 cc and in some embodiments up to about 3000 cc.The volume of fat removed will vary from patient to patient and willdepend on a number of factors including but not limited to: age, bodyhabitus, coagulation profile, hemodynamic stability, severity ofdisease, co-morbidities, and physician preference.

Cells may be administered to a patient in any setting in which abnormalwound healing occurs. The cells may be extracted in advance and storedin a cryopreserved fashion or they may be extracted at or around thetime of defined need. As disclosed herein, the cells may be administeredto the patient, or applied directly to the damaged tissue, or inproximity of the damaged tissue, without further processing or followingadditional procedures to further purify, modify, stimulate, or otherwisechange the cells. For example, the cells obtained from a patient may beadministered to a patient in need thereof without culturing the cellsbefore administering them to the patient. In one embodiment, thecollection of adipose tissue will be performed at a patient's bedside.Hemodynamic monitoring may be used to monitor the patient's clinicalstatus.

In accordance with the invention, the regenerative cells can bedelivered to the patient soon after harvesting the adipose tissue fromthe patient. For example, the cells may be administered immediatelyafter the processing of the adipose tissue to obtain a composition ofregenerative cells. In one embodiment may vary if the cells to bere-infused to the patient are subject to additional modification,purification, stimulation, or other manipulation, as discussed herein.Furthermore, the regenerative cells may be administered multiple times.For example, the cells may be administered continuously over an extendedperiod of time (e.g., hours), or may be administered in multiple bolusinjections extended over a period of time. In certain embodiments, aninitial administration of cells will be administered within about 12hours, such as at 6 hours, and one or more doses of cells will beadministered at 12 hour 30 intervals.

The number of cells administered to a patient may be related to, forexample, the cell yield after adipose tissue processing. A portion ofthe total number of cells may be retained for later use orcyropreserved. In addition, the dose delivered will depend on the routeof delivery of the cells to the patient.

The cells may also be applied with additives to enhance, control, orotherwise direct the intended therapeutic effect. For example, in oneembodiment, and as described herein, the cells may be further purifiedby use of antibody-mediated positive and/or negative cell selection toenrich the cell population to increase efficacy, reduce morbidity, or tofacilitate ease of the procedure. Similarly, cells may be applied with abiocompatible matrix which facilitates in vivo tissue engineering bysupporting and/or directing the fate of the implanted cells. In the sameway, cells may be administered following genetic manipulation such thatthey express gene products that are believed to or are intended topromote the therapeutic response(s) provided by the cells. Examples ofmanipulations include manipulations to control (increase or decrease)expression of factors promoting angiogenesis or vasculogenesis (forexample VEGF). The cells may also be subjected to cell culture on ascaffold material prior to being implanted as described herein.

In one embodiment, direct administration of cells to the site ofintended benefit is preferred. This may be achieved by intravenousinjection. Routes of administration known to one of ordinary skill inthe art, include but are not limited to, subcutaneous, dermal orintramuscular and may or may not include a catheter based mechanism ofdelivery. Cells may be injected in a single bolus, through a slowinfusion, or through a staggered series of applications separated byseveral hours or, provided cells are appropriately stored, several daysor weeks. In one embodiment, the route of delivery will includeintravenous delivery through a standard peripheral intravenous catheteror a central venous catheter. The flow of cells may be controlled byserial inflation/deflation of distal and proximal balloons locatedwithin the patient's vasculature, thereby creating temporary no-flowzones which promote cellular engraftment or cellular therapeutic action.In another embodiment, cells may be resuspended in an artificial ornatural medium or tissue scaffold prior to being administered to thepatient. Systemic therapy would involve injection into the vascularsystem.

Portions of the processed lipoaspirate may be stored before beingadministered to a patient. For short term storage (less than 6 hours)cells may be stored at or below room temperature in a sealed containerwith or without supplementation with a nutrient solution. Medium termstorage (less than 48 hours) is preferably performed at 2-8° C. in anisosmotic, buffered solution (for example Plasmalyte®) in a containercomposed of or coated with a material that prevents cell adhesion.Longer term storage is preferably performed by appropriatecryopreservation and storage of cells under conditions that promoteretention of cellular function, such as disclosed in commonly owned andassigned PCT application number PCT/US02/29207, filed Sep. 13, 2002 andU.S. Provisional application No. 60/322,070, filed Sep. 14, 2001, thecontents of both of which are hereby incorporated by reference.

In accordance with one aspect of the invention, the adipose-tissuederived cells that are administered to a patient can act as growthfactor delivery vehicles. For example, by engineering the cells toexpress one or more growth factors suitable for promoting wound healing,the cells can be administered to a patient, and engineered to releaseone or more of the growth factors. The release can be provided in acontrolled fashion for extended periods of time. For example, therelease can be controlled so that the growth factor(s) are released in apulsed or periodic manner such that there are local elevations in growthfactor, and/or local recessions in the amount of growth factor inproximity to an injured area of tissue.

The cells that are administered to the patient not only help restorefunction to damaged or otherwise unhealthy tissues, but also facilitateremodeling of the damaged tissues. Cell delivery may take place but isnot limited to the following locations: clinic, clinical office,emergency department, hospital ward, intensive care unit, operatingroom, catheterization suites, and radiologic suites. The effects ofcellular therapy can be evident over the course of days to weeks afterthe procedure. However, beneficial effects may be observed as early asseveral hours after the procedure, and may persist for several years.Patients are typically monitored prior to and during the deliver of thecells. Monitoring procedures include, and are not limited to:coagulation studies, oxygen saturation and hemodynamic monitoring. Afterdelivery of cells, patients may require an approximate 24 hour period ofmonitoring for adverse events.

The following examples are provided to demonstrate particular situationsand settings in which this technology may be applied and are notintended to restrict the scope of the invention and the claims includedin this disclosure.

EXAMPLES

The ADC or regenerative cells used throughout the examples set forthbelow can be obtained by the method(s) described in the instantdisclosure and/or the method(s) described in U.S. application Ser. No.10/316,127, entitled SYSTEMS AND METHODS FOR TREATING PATIENTS WITHPROCESSED LIPOASPIRATE CELLS, filed Dec. 9, 2002, which claims priorityto U.S. Provisional Application Serial No. 60/338,856, filed Dec. 7,2001, as well as well as the methods described in U.S. application Ser.No. ______ entitled, SYSTEMS AND METHODS FOR SEPARATING ANDCONCENTRATING REGENERATIVE CELLS FROM TISSUE, filed Jun. 25, 2004, whichclaims priority to U.S. application Ser. No. 10/316,127, entitledSYSTEMS AND METHODS FOR TREATING PATIENTS WITH PROCESSED LIPOASPIRATECELLS, filed Dec. 9, 2002, which are all commonly assigned and thecontents of all of which are expressly incorporated herein by thisreference.

Example 1 Expression of Angiogenic Growth Factor, VEGF, by RegenerativeCells

Vascular Endothelial Growth Factor (VEGF) is one of the key regulatorsof angiogenesis (Nagy et al., 2003; Folkman, 1995). Placenta GrowthFactor, another member of the VEGF family, plays a similar role in bothangiogenesis as well as arteriogenesis. Specifically, transplant ofwild-type (PIGF +/+) cells into a PIGF knockout mouse restores abilityto induce rapid recovery from hind limb ischemia (Scholz et al., 2003).

Given the importance of angiogenesis and arteriogenesis to therevascularization process, PIGF and VEGF expression by the regenerativecells of the present invention was examined using an ELISA assay (R&DSystems, Minneapolis, Minn.) using adipose derived regenerative cellsfrom three donors. One donor had a history of hyperglycemia and Type 2diabetes (a condition highly associated with microvascular andmacrovascular disease). Regenerative cells from each donor were platedat 1,000 cells/cm² in DMEM/F-12 medium supplemented with 10% FCS and 5%HS and grown until confluent. Supernatant samples were taken and assayedfor expression of PIGF and VEGF protein. As shown in FIGS. 16A and 16B,the results demonstrate robust expression of both VEGF (FIG. 16A) andPIGF (FIG. 16B) by the adipose derived regenerative cells of theinvention.

In a separate study, the relative quantity of angiogenic relatedcytokines secreted by cultured regenerative cells from normal adult micewas measured. The regenerative cells were cultured in alpha-MEM with 10%FBS to five days beyond cell confluence, at which time the cell culturemedium was harvested and evaluated by antibody array analysis (RayBio®Mouse Cytokine Antibody Array II (RayBiotech, Inc.). The followingangiogenic related growth factors were detected: Vascular EndothelialGrowth Factor (VEGF), bFGF, Eotaxin, G-CSF, GM-CSF, IL-12 p40/p70, IL-12p′70, IL-13, IL-6, IL-9, Leptin, MCP-1, M-CSF, MIG, PF-4, TIMP-1,TIMP-2, TNF-α, and Thrombopoetin. The following angiogenic relatedgrowth factors or cytokines were elevated at least twice compare toblank control medium with 10% FBS: Vascular Endothelial Growth Factor(VEGF), Eotaxin, G-CSF, IL-6, MCP-1 and PF-4.

These data demonstrate that the regenerative cells of the presentinvention express a wide array of angiogenic and arteriogenic growthfactors. Moreover, the finding that a diabetic patient expressed VEGFand PIGF at equivalent levels to those of normal patients suggest thatdiabetic patients may be candidates for angiogenic therapy by autologousadipose derived regenerative cells.

Example 2 Regenerative Cells Contain Cell Populations that Participatein Angiogenesis

Endothelial cells and their precursors, endothelial progenitor cells(EPCs), are known to participate in angiogenesis. To determine whetherEPCs are present in adipose derived regenerative cells, human adiposederived regenerative cells were evaluated for EPC cell surface markers,e.g., CD-34.

ADCs were isolated by enzymatic digestion of human subcutaneous adiposetissue. ADCs (100 μl) were incubated in phosphate saline buffer (PBS)containing 0.2% fetal bovine serum (FBS), and incubated for 20 to 30minutes at 4° C. with fluorescently labeled antibodies directed towardsthe human endothelial markers CD-31 (differentiated endothelial cellmarker) and CD-34 (EPC marker), as well as human ABCG2 (ATP bindingcassette transporter), which is selectively expressed on multipotentcells. After washing, cells were analyzed on a FACSAria Sorter (BecktonDickenson—Immunocytometry). Data acquisition and analyses were thenperformed by FACSDiva software (BD-Immunocytometry, CA). The results(not shown) showed that the adipose derived regenerative cells from ahealthy adult expressed the EPC marker CD-34 and ABCG2, but not theendothelial cell marker CD-31. Cells expressing the EPC marker CD-34 andABCG2 were detected at similar frequency in regenerative cells derivedfrom a donor with a history of diabetes.

To determine the frequency of EPCs in human adipose derived regenerativecells after their culture in endothelial cell differentiation medium,regenerative cells were plated onto fibronectin-coated plates andcultured in endothelial cell medium for three days to remove matureendothelial cells. Nonadherent cells were removed and re-plated. After14 days, colonies were identified by staining with FITC-conjugated Ulexeuropaeus Agglutinin-1 (Vector Labs, Burlingame, Calif.) and DiI-labeledacetylated LDL (Molecular Probes, Eugene, Oreg.). As shown in FIG. 17,the results indicate an EPC frequency of approximately 500 EPC/10⁶ ADCcells.

The presence of EPCs within the adipose tissue derived regenerativecells indicates that these cells can participate directly in developmentof new blood vessels and enhance angiogenesis and reperfusion.

Example 3 In Vitro Development of Vascular Structures in RegenerativeCells

An art-recognized assay for angiogenesis is one in which endothelialcells grown on a feeder layer of fibroblasts develop a complex networkof CD31-positive tubes reminiscent of a nascent capillary network(Donovan et al., 2001). Since adipose derived regenerative cells containendothelial cells, EPCs and other stromal cell precursors, we tested theability of these regenerative cells to form capillary-like structures inthe absence of a feeder layer. Regenerative cells obtained from inguinalfat pads of normal mice developed capillary networks two weeks afterculture (FIG. 18A). Notably, regenerative cells from hyperglycemic micewith streptozotocin (STZ)-induced Type I diabetes eight weeks followingadministration of STZ formed equivalent capillary networks as thoseformed by cells from normal mice (FIG. 18B).

In a subsequent study, adipose derived regenerative cells were culturedin complete culture medium (α-MEM supplemented with 10% FCS) and noadditional growth factors. These regenerative cells also formedcapillary networks. Furthermore, the vascular structures formed stainedpositive for the endothelial cell markers CD31, CD34, VE-cadherin andvon Willebrand factor/Factor VIII, but not the pan-lymphocyte marker,CD45.

To compare the ability of regenerative cells from young vs. elderly miceto form capillary networks, regenerative cells from normal young andelderly mice (aged 1, 12, or 18 months) were cultured for 2 weeks incomplete culture medium (α-MEM supplemented with 10% FCS) and noadditional growth factors. Equivalent capillary-like networks wereobserved in cultures of regenerative cells from all donors (not shown).

The foregoing data demonstrates that adipose derived regenerative cellsfrom normal and diabetic, as well as young and elderly patients can formvascular structures consistent with the formation of nascent capillarynetworks. Accordingly, the regenerative cells of the invention may beused to treat angiogenic insufficiencies.

Example 4 In Vivo Development of Vascular Structures in RegenerativeCells

In vitro angiogenic potential, while promising, is of little value ifthe cells do not exert in vivo angiogenic activity. Surgically inducinghind limb ischemia is an in vivo model capable of identifying theangiogenic potential of a given therapy. This model was developed inimmunodeficient (NOD-SCID) mice in which the ability of human cells todrive reperfusion could be observed.

Pre-operative blood flow values were determined for both hind limbs asdescribed below. The vasculature of anesthetized mice was tied off witha 4-0 silk ligature at the following sites: 1) iliac artery proximal toits bifurcation, 2) just distal to the origin of deep femoral artery, 3)just proximal to branching of the superficial femoral artery. Afterligation, the vasculature was removed en bloc. Prior to wound closure,grossly observable collaterals branching from the ligated femoralarteries were also ligated. Twenty four hours later, 129S mice wereinjected with 5×10⁶ syngeneic mouse adipose derived regenerative cellsand NOD SCID mice were injected with human adipose derived regenerativecells through the tail vein. Flow was imaged immediately after surgeryand at intervals following treatment using a Laser Doppler Flow Imager(Moor Instruments Inc., Wilmington, Del.). Measurements, taken threetimes per week for 24 days, were normalized to the pre-operative valuefor that limb and presented relative to the control (unoperated) limb.

The model of hind limb ischemia is extremely sensitive to the strain ofmouse used. NOD SCID mice are immunodeficient animals, lacking theability to ignite an acute inflammatory response. For these mice, thissurgical approach generates severe ischemia such that two thirds ofuntreated animals lost hind limb structures below the site of femoralexcision. No cell-treated animal lost any structures above the toe. Yet,for immunocompetent 129S mice, no untreated animals lost any structuresabove the phalanges and displayed an endogenous ability to partiallyregenerate reperfusion. This could be due to the intrinsic angiogenesisassociated with an acute inflammatory response. This may explain whyreperfusion was less extreme when comparing the treated versus controlanimals of different strains.

However, the results showed that mice treated with adipose derivedregenerative cells showed significantly improved perfusion as comparedto untreated mice of both strains. By Day 12, blood flow was restored to50.+−.11% in NOD-SCID mice treated with human regenerative cells, ascompared to 10.+−.10% in untreated mice (p<0.05). Similarly,immunocompetent 129 S mice exhibited 80.+−.12% restoration of flow atday 14, as compared to 56.+−.4% in untreated mice In addition, grossdissection of mice revealed the appearance of collateral vessels in thehind limbs of mice treated with regenerative cells, but not in thosefrom untreated mice or in the healthy limbs of any mice.

Example 5 Increasing ADC Dose is Associated with Improved Graft Survivaland Angiogenesis

Transplant of autologous adipose tissue is a relatively common procedurein plastic and reconstructive surgery {Fulton, 1998; Shiffman, 2001}.However, this procedure is limited by the fact that the adipose tissuefragments are transferred without a vascular supply and, as a result,graft survival is dependent upon neovascularization (Coleman, 1995;Eppley et al., 1990). Thus, in a limited way, the transplanted tissuerepresents an ischemic tissue.

A study in Fisher rats was performed in which adipose tissue fragmentswere transplanted into the subcutaneous space over the muscles of theouter thigh. The right leg was transplanted with 0.2 g of adipose tissuefragments alone, the left leg with 0.2 g of adipose tissue fragmentssupplemented by addition of adipose derived stem cells at threedifferent doses (1.7×10⁵-1.3×10⁶ cells/graft; three animals per dose);in this way the contralateral leg acted as a control. Animals were thenmaintained for one month after which the animals were euthanized and thegrafts recovered, weighed, fixed in formalin and embedded in paraffinfor histologic analysis.

As shown in FIG. 9.1, the results show minimal retention of graftedtissue in the control leg and a dose-dependent increase in retention ofgraft weight in the treated leg. Further, immunohistochemical analysisof the grafts showed considerable neoangiogenesis and perfusion in theadipose derived stem cell treated grafts (FIG. 20B, arrows). This wasalso associated with retention of adipose tissue morphology (FIG. 20B).

Accordingly, Examples 1-5 demonstrate that the adipose derivedregenerative cells of the invention secrete angiogenic and arteriogenicgrowth factors; form nascent capillary networks in vitro; enhancesurvival of fat grafts; and enhance ischemic reperfusion. Thus, theregenerative cells of the invention are capable of promotingangiogenesis and arteriogenesis and may be functional in treatingmultiple diseases with underlying circulatory insufficiencies.

Example 6 Regenerative Cells Secrete Wound Healing Related Cytokines InVitro

Chemokines function in cutaneous wound healing. Recruitment of leukocytesubtypes is tightly regulated by chemokines. Moreover, these chemokinesalso contribute to the regulation of epithelialization, tissueremodeling, and angiogenesis.

Accordingly, the relative quantity of wound healing related cytokinessecreted by cultured ADCs from normal adult mice was measured. ADCs werecultured in alpha-MEM with 10% FBS to five days beyond cell confluence,at which time the cell culture medium was harvested and evaluated byantibody array analysis (RayBio® Mouse Cytokine Antibody Array III(RayBiotech, Inc.).

The results (see Table A) demonstrate that regenerative cells secretegrowth factors and cytokines for neutrophil, macrophage, mast-cell andlymphocyte recruitment in vitro that contribute to re-epithelializationin wound healing process. Specifically, the regenerative cells secreteMIP-1 alpha, RANTES, and MCP-1 that function in macrophage recruitment;MCP-1 that functions in monocyte and mast-cell recruitment; MIG, andTARC that function in lymphocyte recruitment; MIP-2 and KC that functionin wound repair and epithelialization; and TIMP which is ametalloproteinase-1 tissue inhibitor that can reverse unbalancemetalloproteinase in chronic wound to help healing. Macrophage andlymphocytes both produce regulatory and growth promoting factors. Inaddition, mast cells release 11-4, which may stimulate fibroblastactivation.

TABLE A Standard Average Deviation IL-4 316.95 38.9 IL-6 12546.2 339.8Lymphotactin 673.2 6.7 KC 1154.2 65.4 LIX 1825 183.8 MCP-1 17259.41381.7 MCP-5 1638.2 6 MIP-1γ 6632.4 84.1 RANTES 401.7 8.1 SDF-1α 950.753.4 TIMP-1 1485.2 234.4

Example 7 Regenerative Cells Secrete Collagen In Vitro

Regenerative cells were combined with mouse neonatal mouse fibroblastfrom Rosa mouse in three different culture mediums, i.e., DMEM, McCoy's5A and AMEM. Medium was changed twice weekly for 2 weeks. The medium wasnot changed during the third week.

All medium was collected and a collagen assay was performed according tostandard protocol. Briefly, 100 μl supernatant +100 μl 6N HCl, incubateat 100° C. for 16 hours cool and react with Chloramine-T andDimethylaminobenzaldehyde reagent, then detect OD at 550.

No significant difference of collagen secretion can be observed betweenADC and fibroblasts, while there a significant difference can beobserved among different culture mediums. Specifically, in McCoy's 5Amedium, both ADC and fibroblast secrete more collagen than that in DMEMor AMEM (p<0.05). See FIG. 21.

Example 8

Regenerative Cells Accelerate Wound Healing of STZ Treated DiabeticMouse

To determine whether adipose derived regenerative cells can promotewound healing in vivo, 10 mice were treated with STZ for 7 weeks. Atthat time, bilateral 8 mm full thickness skin wounds were created via apunch biopsy. 5 mice were then treated with ADC by local directinjection of 1×10E⁶ (from lacZ donors) per wound in 0.05 ml volume, andthe other 5 were treated with saline and used as a control.

At day 10, the wound size of both groups reached a significantdifference (p=0.002, n=10 wounds, 5 mouse). See FIG. 22. These resultsdemonstrate that adipose derived regenerative cells can aid in mousewound healing.

In addition, to demonstrate engraftment of donor derived cells in thewound, β-galactosidase staining was carried out on the wounds. Theresults (not shown) demonstrated that donor ADC Beta gal-positive cellscan be localized around the injection site, near the capillaries, andclose to wound bed.

Example 9 Regenerative Cells Accelerate Re-Growth of Hair in Mice withWounds

Ten mice were treated with STZ for 7 weeks. After being treated withNair hair remover, bilateral 8 mm full thickness skin wounds werecreated via a punch biopsy. 5 mice were then treated with ADC by localdirect injection at 1×10E⁶ per wound in 0.05 ml volume, and the other 5were treated with saline and used as a control. The identical procedurewas performed on 8 normal B6C57 129 F1 mice, four with ADC localtreatment and 4 with saline treatment as control.

At day 14 the wound size of both experimental groups were measured andimages were taken to record hair recovery rate. The results (not shown)demonstrated that adipose derived regenerative cells not only aided inmouse wound healing, but also significantly accelerated hair grow rate(p<0.05). The histological analysis performed showed donor derivedadipose derived cells around the hair follicle. Thus, ADC can contributeto normal hair restoration simultaneous with promoting wound healing.

A number of publications and patents have been cited hereinabove. Eachof the cited publications and patents are hereby incorporated byreference in their entireties.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. (canceled)
 2. A method of treating an ischemic wound in a subject inneed thereof, comprising: identifying a subject with an ischemic wound;and administering a therapeutically effective amount of a concentratedpopulation of adipose-derived regenerative cells comprising stem cellsto said subject sufficient to accelerate the healing of said ischemicwound.
 3. The method of claim 2, wherein the concentrated population ofadipose-derived regenerative cells comprising stem cells has not beencultured.
 4. The method of claim 2, wherein the concentrated populationof adipose-derived regenerative cells comprising stem cells is obtainedfrom adipose tissue of the subject to whom the cells are administered.5. The method of claim 2, wherein said administering step comprisesapplying the concentrated population of adipose-derived regenerativecells comprising stem cells directly to the ischemic wound.
 6. Themethod of claim 2, wherein the ischemic wound is a diabetic ulcer. 7.The method of claim 2, wherein the subject has peripheral vasculardisease.
 8. The method of claim 2, wherein the subject has ischemicbowel disease.
 9. The method of claim 2, wherein said administering stepcomprises subcutaneous delivery.
 10. The method of claim 2, wherein saidadministering step comprises dermal delivery.
 11. The method of claim 2,wherein the subject is obese.
 12. The method of claim 2, wherein theischemic wound is an ischemic stroke.
 13. The method of claim 2, whereinthe ischemic wound is an ischemic cardiomyopathy.
 14. The method ofclaim 2, wherein the subject has rheumatoid arthritis.
 15. The method ofclaim 2, wherein the subject has lymphedema.
 16. The method of claim 2,wherein said administration step comprises administering multiple dosesof said concentrated population of adipose-derived regenerative cellscomprising stem cells.
 17. The method of claim 2, further comprising:providing adipose tissue to a self-contained adipose tissue processingunit comprising: a tissue collecting container configured to receive andfully or partially disaggregate adipose tissue, wherein said tissuecollecting container is coupled to a cell collection container whereinsaid coupling defines a closed system in which said fully or partiallydisaggregated adipose tissue directed from tissue collecting containerto cell collection container is not exposed to the external environment;and wherein said cell collection container, further comprises a cellconcentrator that facilitates separation of the cells in a suspension;and an outlet that is structured to permit said concentrated populationof cells comprising adipose-derived cells to be removed from saidadipose tissue processing unit; and obtaining the concentratedpopulation of cells comprising adipose-derived regenerative cells fromsaid self-contained adipose tissue processing unit.
 18. The method ofclaim 2, wherein the therapeutically effective amount of theconcentrated population of adipose-derived regenerative cells comprisesa minimum concentration of between 1×10⁵ and 1×10⁷ adipose-derived stemcells.
 19. The method of claim 2, wherein the concentrated population ofadipose-derived regenerative cells comprising stem cells furthercomprises an additive.
 20. The method of claim 17, wherein said additiveis selected from the group consisting of a cell differentiation factor,a growth promoter, an immunosuppressive agent, and a medical device, ora combination thereof.
 21. The method of claim 17, wherein said additiveis selected from the group consisting of other cells, tissue, and tissuefragments.
 22. The method of claim 17, wherein said additive comprisesan angiogenic factor, or an arteriogenic factor.
 23. The method of claim2, wherein said concentrated population of adipose-derived regenerativecells comprising stem cells further comprises endothelial precursorcells.
 24. The method of claim 2, wherein said concentrated populationof adipose-derived regenerative cells comprising stem cells is appliedto a biocompatible matrix.
 25. The method of claim 2, wherein saidconcentrated population of adipose-derived regenerative cells comprisingstem cells is applied to a bandage.
 26. The method of claim 2, whereinsaid concentrated population of adipose-derived regenerative cellscomprising stem cells in not exposed to the external environment priorto the administration step.